bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolism of 2‐Deoxy‐D‐galactose in Liver Induces Phosphate and Uridylate Trapping
Authors: STARLING James J. , KEPPLER Dietrich O. R.
Published: March 2005

The metabolites of 2‐deoxy‐D‐galactose were quantitatively determined in liver and in various tissues of the rat. After intraperitoneal administration of 2‐deoxy‐D‐[1‐14C]galactose in a dose of 1 mmol/kg the liver was the predominant organ metabolizing this galactose analog. 2‐Deoxy‐D‐galactose 1‐phosphate and UDP‐2‐deoxy‐D‐galactose were the major hepatic metabolites and after 3 h comprised 78% and 20% of the total radioactivity in liver, respectively; UDP‐2‐deoxy‐D‐glucose amounted to 0.7%. Liver was the only tissue in which substantial formation of UDP‐2‐deoxy‐D‐hexoses was observed.

The synthesis of UDP‐2‐deoxy‐D‐galactose was shown to be catalyzed by UDP‐glucose:galactose‐1‐phosphate uridylyltransferase. The low affinity of this enzyme for 2‐deoxy‐D‐galactose 1‐phosphate explained the accumulation of this substrate analog to levels as high as 14 mmol/kg liver. Enzymatic analysis of alkaline ethanol extracts showed a marked fall of the hepatic Pi content during the period of rapid 2‐deoxy‐D‐galactose 1‐phosphate formation. The trapping of Pi was associated with a loss of ATP and a lowering of the adenylate energy charge.

The formation of UDP‐2‐deoxy‐D‐galactose occurred only after high levels of 2‐deoxy‐D‐galactose 1‐phosphate had been attained. The limited epimerization of UDP‐2‐deoxy‐D‐galactose to UDP‐2‐deoxy‐D‐glucose led to an accumulation of the former deoxy‐sugar nucleotide. The trapping of uridylate in the form of UDP‐2‐deoxy‐D‐galactose was accompanied by a transient decrease in the hepatic contents of UTP, UDP‐glucose, and UDP‐galactose. The administration of 2‐deoxy‐D‐galactose together with uridine or orotate completely prevented the depression of UTP.

The accumulation of 2‐deoxy‐D‐galactose 1‐phosphate and the associated depression of Pi, in the absence of UTP deficiency, represent a state analogous to that induced by galactose in uridylyltransferase‐deficient cells and tissues and may serve as a biochemical model in the study of the cellular injury in galactosemia.
The Distribution of Lipids in the Protoplast Membranes of Bacillus subtilis
Authors: BISHOP David G. , OP DEN KAMP Jos A. F. , VAN DEENEN Laurens L. M.
Published: March 2005

The distribution of phospholipids in the protoplast membrane of Bacillus subtilis has been studied by comparing the effects of treatment with phospholipase C or trinitrobenzenesulphonic acid on intact protoplasts or membranes derived from them. After 2 h treatment of protoplasts at 37°C with phospholipase C (B. cereus), 45–50% of the membrane phospholipid has been hydrolyzed. including 90% of the phosphatidylethanolamine and 70–80% of the lysylphosphatidylglycerol. but no lysis of the protoplasts can be detected. Complete hydrolysis of phosphatidylethanolamine and lysylphosphatidylglycerol by phospholipase C is obtained in isolated membranes at a rate greater than in intact protoplasts. The rate and extent of hydrolysis by phospholipase C in both protoplasts and membranes is strongly temperature‐dependent and part of this effect is ascribed to phase separations in the membrane lipid reducing accessibility of the substrates.

About 60% of the aminophospholipids in intact protoplasts are labelled by exposure to trinitrobenzenesulphonic acid and this level increases to only 70% in isolated membranes. It is concluded that the introduction of the bulky head group into both protein and lipid during trinitrobenzenesulphonic acid labelling precludes the reaction proceeding to completion.

The results do not permit an unequivocal distribution of the phospholipids between the inner and outer halves of the bilayer. It appears however that at least 60% of the phosphatidylethanolamine is located in the outer monolayer, and under conditions where lysylphosphatidylglycerol is accumulated, that at least 60% of this lipid is also in the outer monolayer. Furthermore, the membrane appears to contain the capacity for transbilayer movement (flip‐flop) of at least the aminophospholipids and this capacity is expressed when the composition of the outer half of the bilayer is modified by treatment with phospholipase C.
An Analysis of the Contracted Sheath Structure of Bacteriophage Mu
Authors: CREMERS Alfons F. M. , SCHEPMAN A. Marius H. , VISSER Marja P. , MELLEMA Jan E.
Published: March 2005

Polymers of contracted sheath particles of bacteriophage Mu were imaged by the negativestaining technique and the electron images were analyzed by optical and digital methods.

By means of the three‐dimensional reconstruction technique of DeRosier and Klug [Nature (Lond.) 217, 130–134 (1968)] an averaged density map of the sheath structure at a resolution of about 2.0 nm was derived. The sheath is known to consist of one type of protein with a molecular weight of about 52000 [Admiraal and Mellema, J. Ultrastr. Res. 56, 48–64(1976)].

The interpretation of the map has given information about packing and shape of the protein subunits. One way to describe the structure is by a set of annular rings with 6‐fold symmetry. The height of these rings is about 1.8 nm and successive rings in the structure change by about 33° in azimuth. The protein subunit which occupies more than one ring in the polymer, is elongated. The long axis of the protein subunit is at an angle of about 20° with the plane normal to the polymer axis. These data are discussed in relation to changes in the sheath molecules upon contraction.
6‐Aminohexanoic Acid Cyclic Dimer Hydrolase. A New Cyclic Amide Hydrolase Produced by Acromobacter guttatus KI72
Authors: KINOSHITA Shinichi , NEGORO Seiji , MURAMATSU Minoru , BISARIA V. S. , SAWADA Shujo , OKADA Hirosuke
Published: March 2005

6‐Aminohexanoic acid cyclic dimer hydrolase produced by Achromobacter guttatus cells grown on 6‐aminohexanoic acid cyclic dimer (1,8‐diazacyclotetradecane‐2,9‐dione) was purified until it was homogeneous according to polyacrylamide disc gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis, Sephadex G‐200 column chromatography, and analytical centrifugation. The following characteristics of the purified enzyme were observed.

(1) The molecular weight of the enzyme, estimated to be 110 000 by column chromatography on Sephadex G‐200 and 100 000 by sedimentative equilibrium, together with the minimum molecular weight of 55 000 obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicated it is a dimeric enzyme. The sedimentation constant (s20) was estimated to be 5.7 S from the sedimentation velocity.

(2) The enzyme had an optimum pH of 7.3 when 6‐aminohexanoic acid cyclic dimer was used as substrate. It was stable in a pH range between 5.5 to 8.5, and 50% and 100% of activity was lost after 10 min of heating at 45°C and 50°C respectively. The optimum temperature for activity was 33°C. The Michaelis‐Menten constant (Km) was 6 mM toward the dimer, and the turnover number was 8 s−1 assuming a molecular weight of 100 000.

(3) This enzyme was strongly inhibited by 1 μM diisoprpylphosphofluoridate and 10 μM p‐chloromercuribenzoate but scarcely inhibited by 100 mM ethylenediaminetetraacetic acid. The enzyme inhibited by p‐chloromercuribenzoate could be reactivated by 2‐mercaptoethanol.

(4) This enzyme was only active on 6‐aminohexanoic acid cyclic dimer to form 6‐aminohexanoyl‐6‐aminohexanoic acid. It was inactive on 6‐aminohexanoic acid oligomers (degree of polymerization 2 to 6), various cyclic amides, cyclic diamides, amides, oligopeptides, and casein.

(5) The absorption coefficient (A1%280 nm) of the enzyme was 19.4. The enzyme was composed of 500 amino acid residues including 3 cysteines; from this the molecular weight was calculated to be 103 200 as a dimer.

(6) 6‐Aminohexanoic acid cyclic dimer hydrolase was classified as a new cyclic amide hydrolase.
Human α‐N‐Acetylglucosaminidase
Authors: von FIGURA Kurt
Published: March 2005

Purified urinary α‐N‐acetylglucosaminidase acts as an exoglycosidase. The enzyme removes from heparan sulfate exclusively α‐glycosidically linked N‐acetylglucosamine residues. The pH optimum of around 4.4 towards heparan sulfate and heparin is similar to that towards synthetic arylglycosides. Urinary α‐N‐acetylglucosaminidase can be separated by isoelectric focusing into multiple forms with pI values between 3.3 and 6.0. The multiple forms differ in their recognition and endocytosis by cultivated skin fibroblasts. Forms with pI values of 4.8 ± 0.3 respond best to endcytosis. From these forms up to 0.8 × 106 molecules may be recognized and taken up in an hour by a single cell. Sodium periodate treatment reduces the α‐N‐acetylglucosaminidase recognition by fibroblasts and suggests that the recognition sites on the enzyme are associated with its carbohydrate moiety. Attempts to modify the recognition of α‐N‐acetylglucosaminidase by pretreatment with purified glycosidases failed.
Effect of Borate on the Catalytic Activities of Muscle Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: WOLNY Marian
Published: March 2005

The effect of borate on glyceraldehyde‐3‐phosphate dehydrogenase from human, pig and rabbit muscle was studied. At lower concentration of borate only the dehydrogenase activity is inhibited, reversibly and competitively against NAD. At concentration of borate above 6 mM the plots of 1/vversus borate concentration become nonlinear and the inhibition is extended to the esterase and acetylphosphatase activities. In certain conditions a time‐dependent inactivation and reactivation was observed. The direct interaction between borate (if present at concentration of at least 6 mM) and glyceraldehyde‐3‐phosphate dehydrogenase is postulated, the possible site of the reaction being the histidine residue(s).

The esterase activity of the human muscle enzyme and the effect of borate on it are different from the other mammalian enzymes.
The Replication of Ribosomal DNA in Physarum polycephalum
Authors: VOGT Volker M. , BRAUN Richard
Published: March 2005

The DNA coding for ribosomal RNA in Physarum polycephalum exists as a collection of extrachromosomal molecules of molecular weight 37 × 106. We have investigated the replication of rDNA, with the following results. (a) Replication of rDNA is unscheduled. This means that molecules that are replicated at any particular time in one mitotic cycle have an equal probability of replicating again in each time interval in the subsequent cycle. Similarly, in a single cycle, some molecules replicate more than once, and some not at all. (b) Replication forks appear to move bidirectionally from points 45% or 33% from one end of the DNA. Replicating molecules observed by electron microscopy are all linear.
Thyrotropin Stimulation of a Thyroid Contractile Protein Phosphorylation
Authors: LAMY Françoise , LECOCQ Raymond , HAUMONT Michèle , DUMONT Jacques E.
Published: March 2005

Contractile proteins were isolated from preincubated dog thyroid slices by a procedure identical to the one used for the preparatio of muscle actomyosin. At least two of these proteins (molecular weight = 26000 and 15000) were phosphorylated in the intact cell system. After one hour of incubation of the slices with thyrotropin (10 mU/ml) and [32P]phosphate, the specific activity of the 26000‐Mr protein was increased by a factor of three while no significant change in specific activity was observed in the 15000‐Mr contractile protein. This effect of thyrotropin was already observed after 30 min of action, was elicited at hormone concentrations of the same order as those required to induce secretion and has been reproduced by dibutyryl adenosine 3′:5′‐monophosphate.

Cycloheximide (0.35 mM) which almost totally inhibited protein synthesis in thyroid slices. did not affect the thyrotropinstimulation of the phosphorylation of the 26000‐Mr protein.

The phosphorylation of serine residue(s) has been demonstrated in this protein but the presence of radioactive phosphothreonine could not be detected.

On the basis of its molecular weight, the 26000‐Mr protein could be similar to the inhibitory component of muscle troponin complex (TN‐I).
Non‐equilibrium Method for the Radioimmunoassay of Clozapine in the Presence of Metabolites
Authors: ROSENTHALER Joachim , NIMMERFALL Fritz , SIGRIST Robert , MUNZER Hans
Published: March 2005

Cross‐reactions with metabolites are an ever‐recurring problem encountered in the use of radioimmunoassay techniques to determine active compounds in biological material. Metabolites may interfere with the assay of the parent drug to a variable extent. Taking 8‐chloro‐11‐(4‐methyl‐1‐piperazinyl)‐5H‐dibenzo[b,e][1,4]d iazepine (clozapine) as an example, it was shown that the extent to which the antiserum produced interacts with the parent drug and the metabolites can be estimated by determining the equilibrium constants and the kinetics.

In the present case, therefore, it was advantageous to carry out the radioimmunoassay in disequilibrium, i.e, in order to differentiate the metabolites from the parent drug. the sample was incubated with the antiserum for 10 min, after which the labelled antigen was added and the reaction mixture again incubated for a brief, exactly timed interval. It was shown that cross‐reactions did not occur in mixtures of clozapine and its N‐demethyl and N‐oxide metabolites in the proportions 1: 1: 2 over a range of concentration of 1.5–48 ng clozapine per 100 μl human plasma. The equilibrium constants measured with the clozapine goat antiserum were as follows: clozapine 1.2 × 108, M−1, the N‐demethyl metabolite 4.6 × 107 M−1 and the N‐oxide metabolite 3.7 × 107 M−1 (pH 7.5 and 20 °C).
The Hyphal Wall of Mucor mucedo
Authors: DATEMA Roelf , VAN DEN ENDE Herman , WESSELS Joseph G. H.
Published: March 2005

Treatment of isolated hyphal walls of Mucor mucedo with nitrous acid resulted in the release of two water‐soluble polyanions: (a) a glycuronan, containing all the neutral sugars and uronic acid present in the hyphal wall and (b) an inorganic polyphosphate. The glycuronan was purified and some of its properties were studied. The glycuronan could also be extracted quantitatively with salt solutions of high ionic strength and partially with a solution of potassium hydroxide. This is presented as evidence that the glycuronan is a genuine constituent of the cell wall, non‐covalently bound to glucosamine‐containing polymers which are susceptible to depolymerization by nitrous acid. By treatment with acid the glycuronan was partly converted to crystalline poly(glucuronic acid) with the properties of mucoric acid. This strongly suggests that mucoric acid, which can be extracted from the walls of M. mucedo by alkali after acid treatment, is not a genuine wall component but arises by partial acid hydrolysis of the heteropolymeric glycuronan.
Specificity of Sialyltransferase: Structure of α1‐Acid Glycoprotein Sialylated in vitro
Authors: VAN DEN EIJNDEN Dirk H. , STOFFYN Pierre , STOFFYN Anne , SCHIPHORST Wietske E. C. M.
Published: March 2005

The terminal galactosyl units of desialylated α1‐acid glycoprotein were selectively labeled with tritium by a galactose oxidase/NaB3H4 procedure. The 3H‐labeled glycoprotein was effective as an acceptor in sialyltransferase reactions catalyzed by rat liver microsomes in vitro with unlabeled CMP‐N‐acetyl‐neuramininic acid as sialic acid donor. Permethylation/hydrolysis of glycopeptides derived from the resialylated 3H‐labeled glycoprotein yielded radioactive 2,3,4‐trimethylgalactose indicating that rat liver microsomes are capable of transferring sialic acid to position C‐6 of the terminal galactosyl units of desialylated α1‐acid glycoprotein. No indication was obtained for transfer of sialic acid to other positions.

This result is discussed in view of the multiplicity of positions of attachment of sialic acid to galactosyl residues in native α1‐acid glycoprotein.
The Peptidoglycan Crosslinking Enzyme System in Streptomyces Strains R61, K15 and rimosus
Authors: LEYH‐BOUILLE Mélina , DUSART Jean , NGUYEN‐DISTÈCHE Martine , GHUYSEN Jean‐Marie , REYNOLDS Peter E. , PERKINS Harold R.
Published: March 2005

The dd‐carboxypeptidase‐transpeptidase enzyme system in Streptomyces strain K15 consists of: (1) a membrane‐bound transpeptidase capable of performing low dd‐carboxypeptidase activity; and (2) a set of dd‐carboxypeptidases: (a) membrane‐bound, (b) lysozyme‐releasable and (c) exocellular, having low transpeptidase activities in aqueous media and at low acceptor concentrations. The dd‐carboxypeptidases are related to each other and may belong to the same pathway leading to enzyme excretion. A similar enzyme system occurs in Streptomyces strain R61 except that the membrane‐bound dd‐carboxypeptidase activity is low when compared with the membrane‐bound transpeptidase activity. In Streptomyces rimosus the enzyme system consists almost exclusively of the membrane‐bound transpeptidase and the levels of membrane‐bound, lysozyme‐releasable and exocellular dd‐carboxypeptidases are very low.
The Peptidoglycan Crosslinking Enzyme System in Streptomyces R61, K15 and rimosus. Immunological Studies
Authors: NGUYEN‐DISTÈCHE Martine , FRÈRE Jean‐Marie , DUSART Jean , LEYH‐BOUILLE Mélina , GHUYSEN Jean‐Marie , POLLOCK Jerry J. , IACONO Vincent J.
Published: March 2005

The exocellular dd‐carboxypeptidases from Streptomyces R61, K15, the lysozyme‐releasable dd‐carboxypeptidases from Streptomyces R61, K15 and rimosus, and the membrane‐bound dd‐carboxypeptidase of Streptomyces K15 are immunologically related to each other.
Aspects on Microenvironmental Compartmentation
Authors: KOCH‐SCHMIDT Ann‐Christin , MATTIASSON Bo , MOSBACH Klaus
Published: March 2005

Soluble bifunctional enzyme aggregates have been prepared by cross‐linking the sequential enzymes malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 4.1.3.7) using glutaraldehyde. The kinetic behaviour of this two‐enzyme system in its aggregated and non‐aggregated form was studied both in free solution and immobilized on Sepharose beads. This study was undertaken in order to distinguish between the following two factors which may account for the increased efficiency found in general in co‐immobilized consecutive two‐enzyme systems:

closer proximity between the participating enzymes and

establishment of a favourable microenvironment (such as higher local intermediate concentration caused by increased diffusional hindrance in the gel phase).

It was found that in spite of a reduction of the distance between the two enzymes in the aggregated form by an estimated factor of 103, no kinetic advantage (shorter lag phase or higher steady‐state rate) could be detected compared to the corresponding system with the two enzymes not linked to each other. However, both systems immobilized to Sepharose reached the steady‐state rate of citrate formation almost immediately, in contrast to the corresponding free systems which exhibited pronounced lag phase.

These results indicate that, at least in the above systems and under the conditions given, diffusional hindrance in the gel phase of the intermediate oxaloacetate, which is present in rate‐limiting concentrations, is the dominant cause of the observed higher efficiency in immobilized systems.
Purification and Properties of Glycogen Synthase I from Human Leukocytes
Authors: SØLLING Henrik , ESMANN Viggo
Published: March 2005

Glycogen synthase I was purified from human polymorphonuclear leukocytes by a procedure involving affinity chromatography of the glycogen‐enzyme complex, digestion of endogenous glycogen by amylase, starch chromatography and gel filtration. The purified enzyme had a specific activity of 7–11 U/mg protein, or 4–5 U when expressed per mg of residual glycogen. Further purification to 21 U/mg protein could be achieved. The enzyme was inactive in the absence of added glycogen. A subunit molecular weight of 85000 was determined by polyacrylamide electrophoresis in sodium dodecylsulfate. The molecular weight of the native enzyme was estimated to be 390000 (13.2 S) by sucrose gradient centrifugation and 410000 by gel filtration indicating that the native enzyme is a tetramer. The gel filtration behavior was not affected by enzyme concentration, temperature, or the presence of ligands. The energy of activation was estimated to 13500 cal/mol (56.5 kJ/mol), corresponding to a Q10of 2.2. In the presence of glucose 6‐phosphate or Na2SO4, the enzyme showed a broad pH optimum between pH 6.8–9.2. In the absence of these ligands and in particularly in the presence of Mg2+, the enzyme is sensitive to small changes of pH in the interval pH 7.4–8.4.

During purification, synthase I requires protection by 0.6 mM dithiothreitol, while high concentrations of mercaptoethanol or dithiothreitol inactivates the enzyme, particularly during freezing. During 24‐h incubations, synthase I undergoes a spontaneous, temperature‐dependent inactivation which is not due to proteolysis, but presumably is caused by irreversible conformational changes. These can be prevented by high concentrations of glucose 6‐phosphate, Na2SO4, inorganic phosphate, UDP and glycogen. Mg2+and traces of ethanol inactivates the enzyme. The lyophilized enzyme is stable for years.
Structure of Seven Oligosaccharides Excreted in the Urine of a Patient with Sandhoff's Disease (GM2 Gangliosidosis‐Variant O)
Authors: STRECKER Gérard , HERLANT‐PEERS Marie‐Claire , FOURNET Bernard , MONTREUIL Jean , DORLAND Lambertus , HAVERKAMP Johan , VLIEGENTHART Johannes F. G. , FARRIAUX Jean‐Pierre
Published: March 2005

The urine of a patient with Sandhoff's disease (GM2 gangliosidosis‐variant O) contains 10–12 N‐acetylglucosamine‐rich oligosaccharides in high amounts. The structures of seven of these have been determined: β‐GlcNAc‐(1→2)‐α‐Man‐(1→3)‐β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→4)‐α‐Man‐(1→3)‐β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→2)‐α‐Man‐(1→6)‐β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→4)‐α‐Man‐(1→6)‐β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→2)‐α‐Man‐(1→3)‐[β‐GlcNAc‐(1→2)‐α‐Man‐(1→6)]β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→2)‐α‐Man‐(1→3)[β‐GlcNAc‐(1→2)‐α‐Man‐(1→6)] β‐GlcNAc‐(1→4)]β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→2)‐α‐Man(1)‐(1→3) [β‐GlcNAc‐(1→2)‐α‐Man(2)‐(1→6)]β‐Man‐(1→4)‐GlcNAc, with additional β‐GlcNAc‐(1→4) on mannose (1) or (2).

An unusual oligosaccharide, with a tri‐branched β‐mannose, has been characterized as the major component excreted in urine.
Purification and Characterization of Two Aldehyde Dehydrogenases from Pseudomonas aeruginosa
Authors: GUERRILLOT Luc , VANDECASTEELE Jean‐Paul
Published: March 2005

Two soluble aldehyde dehydrogenases isoenzymes have been purified and separated from extracts of a paraffin‐assimilating bacterium, Pseudomonas aeruginosa. The first one, obtained at an estimated purity of 20% (spec. act. with butanal 0.33 kat/kg) was NAD‐dependent. It was rapidly inactivated at pH 8.6 but was efficiently protected by NAD. It had a molecular weight of 225000 and presented a high affinity for aldehydes of short and middle chain lengths. The second enzyme, obtained in a nearly homogenous state (spec. act. with pentanal 0.62 kat/kg) was NADP‐dependent. It was activated by ions, in particular potassium ions, and had a good affinity for aldehydes of higher chain lengths. Both enzymes were stabilized by thiols and glycerol and were inactivated by reagents of sulfhydryl groups. These enzymes are ‘constitutive’ and their physiological function is uncertain. When the bacteria were grown on n‐paraffin a new membrane‐bound NAD‐dependent aldehyde dehydrogenase activity was produced.
A 31P‐Nuclear‐Magnetic‐Resonance Study of the Phosphate Groups in Lipopolysaccharide and Lipid A from Salmonella
Authors: MÜHLRADT Peter F. , WRAY Victor , LEHMANN Volker
Published: March 2005

Untreated and partially deacylated lipopolysaccharides from various P− and P+ strains of Salmonella were studied with 31P nuclear magnetic resonance spectroscopy and by conventional analytical methods.

The spectral signals were assigned to various phosphate groups in the lipid A moiety and in the oligosaccharide part. A signal at + 2.3 ppm could be assigned to a phosphodiester linkage formed between 4‐amino‐4‐deoxy‐l‐arabinose linked via the glycosidic hydroxyl group to the 4′‐phosphate group of the glucosamine disaccharide in the lipid A moiety. A strong pyrophosphate signal at + 11 ppm in P− strains was identified as a pyrophosphoryl ethanolamine group at the glycosidic end of this glucosamine disaccharide unit. No evidence was found for phosphodiester or pyrophosphodiester bonds crosslinking lipopolysaccharide ‘subunits’. A revised version of the lipid A structure of Salmonella is presented.

By a combination of 31P nuclear magnetic resonance spectroscopy data and conventional analytical methods the extent to which the lipopolysaccharides are substituted by various phosphate groups on the lipid A and the oligosaccharide moiety could be estimated. It was thus shown that substantial heterogeneity, leading to several molecular species of lipopolysaccharides is caused by addition or omission of certain groups. Since changes in substitution were found to be dependent on the growth conditions, it is thought possible that the overall negative surface charge of Salmonella can be modified by addition or omission of neutralising amino groups from ethanolamine and/or 4‐amino‐4‐deoxy‐l‐arabinose, and can thus be adapted to the environment.
Identity of DNA Polymerase γ from Synaptosomal Mitochondria and Rat‐Brain Nuclei
Authors: HÜBSCHER Ulrich , KUENZLE Clive C. , SPADARI Silvio
Published: March 2005

DNA polymerase γ and mitochondrial DNA polymerase were isolated from brain nuclei and synaptosomes respectively. The presence of a single DNA polymerase in synaptosomal mitochondria was established by chromatography on DEAE‐cellulose, phosphocellulose and DNA‐cellulose, as well as by sedimentation analysis and isoelectric focusing. A great similarity between the purified nuclear DNA polymerase γ and the mitochondrial enzyme was found by the following criteria: chromatographic behaviour in three column systems; essentially complete inhibition by N‐ethylmaleimide (2 mM); optimal requirements for Mn2+ (0.1 mM), Mg2+ (5 mM) and pH (8.0); template preferences, poly(A) · (dT)20–25 > activated DNA > poly(dA) · (dT)12–18; lack of activity on single‐stranded polynucleotides and (dT)12‐primed mRNA; molecular weight (180 000), sedimentation (9.2 S) and isoelectric point (pI5.4). We therefore conclude that brain nuclear DNA polymerase 7 and synaptosomal mitochondrial DNA polymerase are closely related and may even be identical.
Stereochemistry of Olefinic Bond Formation in Defensive Steroids of Acilius sulcatus (Dytiscidae)
Authors: CHAPMAN John C. , LOCKLEY William J. S. , REES Huw H. , GOODWIN Trevor W.
Published: March 2005

The defensive secretion of Acilius sulcatus contains a number of pregnane derivatives : cortexone, 20α‐hydroxy‐4‐pregnen‐3‐one, together with the unusual Δ4,6 dienes, 6,7‐dehydrocortexone, 20α‐hydroxy‐4,6‐pregnadien‐3‐one and 4,6‐pregnadien‐3,20‐dione. The synthesis of all these steroids except cortexone is described. Complete separation of the steroids of Acilius can be achieved by high‐performance liquid chromatography on a reversed‐phase column system. During biosynthesis of the Acilius steroids from cholesterol, introduction of the Δ4 and Δ6 bonds involves elimination of the 4β and 7β hydrogens, respectively. Possible mechanisms of formation of the Δ4,6 steroids are discussed.
Intermediates in the Conversion of Procollagen to Collagen
Authors: DAVIDSON Jeffrey M. , McENEANY Linda S. G. , BORNSTEIN Paul
Published: March 2005

Intermediates in the conversion of procollagen to collagen were isolated from radioactively labeled chick cranial bones by ion‐exchange chromatography. Cleavage of these proteins with vertebrate collagenase revealed that each of the several forms of these intermediates lacked NH2‐terminal but retained COOH‐terminal extensions. The chain composition of each intermediate was resolved by two‐dimensional slab gel electrophoresis. The intermediates differed from each other in having sustained cleavages in zero, one or two pcα chains. The relative proportions of intermediates with different intact pcα chains, observed in conversion of procollagen, have enabled us to construct a detailed model of the stepwise limited proteolysis of procollagen.
Nuclear‐Magnetic‐Resonance Study of the Histidine Residues of S‐Peptide and S‐Protein and Kinetics of 1H‐2H Exchange of Ribonuclease A
Authors: BRADBURY J. Howard , CROMPTON Malcolm W. , TEH Joo Seng
Published: March 2005

1H NMR spectroscopy at 100 MHz was used to determine the first‐order rate constants for the 1H‐2H exchange of the H‐2 histidine resonances of RNase‐A in 2H2O at 35°C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35°C and pH meter reading 11 caused irreversible denaturation of RNase‐A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H‐3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem. (Tokyo) 75, 1197–1200 1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed.

The first‐order rate constant for the exchange of His‐48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5. The corresponding values for His‐119 show a decrease and those for His‐12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase‐A (probably due to the titration of Tyr‐25) which allows His‐48 to become accessible to solvent.

1H NMR spectra of S‐protein and S‐peptide, and of material partially deuterated at the C‐2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase‐A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936–937]. The chemical shifts of the C‐2 and C‐4 protons of histidine‐12 of S‐peptide are followed as a function of pH and a pK′ value of 6.75 is obtained.

The reassignment of the three C‐2 histidine resonances of S‐protein is confirmed by partial deuteration studies. The pK′ values obtained from titration of the H‐2 resonances of His‐48, His‐105 and His‐119 are 5.3, 6.5 and 6.0, respectively. The S‐protein is less stable to acid than RNase‐A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26°C. His‐48 in S‐protein titrates normally and has a lower pK than in RNase‐A probably because of the absence of Asp‐14, which in RNase‐A forms a hydrogen bond with His‐48 and causes it to be inaccessible to solvent, at pH values below 9.
On the Mechanism of Substrate Binding to the Purine‐Transport System of Saccharomyces cerevisiae
Authors: FORÊT Martine , SCHMIDT Rainer , REICHERT Uwe
Published: March 2005

The yeast Saccharomyces cerevisiae takes up adenine, guanine, hypoxanthine, and cytosine via a common energy‐dependent transport system. The apparent affinity of the transport system to these and other purines and pyrimidines is correlated with their capability to be protonated to the positively charged form. Further organic molecules are competitive inhibitors when they are cationic, e.g. guanidine and octylguanidine in contrast to urea, or hexadecyltrimethylammonium in contrast to dodecylsulfate and Triton X‐100.

The influence of the pH on the kinetic constants of hypoxanthine transport points to a stoichiometry of one proton being associated to the transport system together with one substrate molecule.

The pKa values of two ionizable groups that are involved in substrate binding are revealed; one of which (pKa= 1.8) may be attributed to the substrate, the other (pKa= 5.1) to an amino acid residue in the recognition site of the transport system. Studies with group‐specific inhibitors indicate that this amino acid residue contains a carboxyl group.

The results are in accordance with the assumption that a carboxyl group of the transport system, a proton and a substrate molecule arrange to an uncharged ternary complex.
The Structure of Human‐Plasma Low‐Density Lipoprotein B
Authors: MÜLLER Karl , LAGGNER Peter , GLATTER Otto , KOSTNER Gerhard
Published: March 2005

The X‐ray small‐angle scattering of human plasma lipoprotein B of the low‐density fraction (ϱ= 1.016−1.060 g · cm−3) has been recorded to high precision at different electron density contrasts.

The overall structure of the particles is characterized by a quasi‐spherical shape and radial symmetry. A maximum diameter of 23 nm and a molecular weight of 2.4 × 106 have been determined.

The internal structure is described in terms of a model consisting of spherical layers with different electron densities indicating that the neutral lipids are arranged in the core of the molecule up to a radius of about 8 nm surrounded by a monolayer of free cholesterol, phospholipids and protein. The neutral lipids are shown to be in an ordered, liquid crystalline state at 4°C and to undergo a thermotropic transition into a disordered state at higher temperatures.
Effect of Methylxanthines on Binding of the Glucocorticoid Receptor to DNA‐Cellulose and Nuclei
Authors: CAKE Max H. , LITWACK Gerald
Published: March 2005

The binding of [3H]dexamethasone·receptor complex from rat liver cytosol to isolated nuclei or DNA‐cellulose can be greatly enhanced at low temperature by the presence of theophylline. Aminophylline and caffeine can mimic this effect; however, papaverine and 1‐methyl‐3‐isobutylxanthine, at concentrations inhibitory to phosphodiesterase, are without effect on glucocorticoid receptor binding to DNA. Furthermore, theophylline can be added when adenosine 3′:5′‐monophosphate‐(cAMP) hydrolysis is already complete and still enhance DNA binding. These results imply that this effect of theophylline is independent of its known effect on cAMP levels. Activation by methyl‐xanthines does not alter the sedimentation of the glucocorticoid·receptor complex in sucrose gradients but does alter the pI and in this respect brings about changes resembling those which occur upon activation by heat. Recently we have shown that pyridoxal phosphate inhibits the binding of heat‐activated receptor to DNA‐cellulose. Similarly, we have shown here that pyridoxal phosphate also inhibits the DNA‐cellulose binding of theophylline‐treated receptor. The presence of theophylline also enhances the rate of binding of [3H]dexamethasone to the receptor and increases its apparent affinity for the steroid. The data suggest that the effect of theophylline is on some cytosolic component, perhaps the receptor itself. Enhanced DNA binding as a result of exposure to theophylline at low temperature can also be demonstrated using the glucocorticoid receptor of kidney, thymus and Reuber H35 cells.
Enzyme Leakage and Multipoint Attachment of Agarose‐Bound Enzyme Preparations
Authors: LASCH Jürgen , KOELSCH Regine
Published: March 2005

The solvolytic detachment of leucine aminopeptidase fromSepharose‐enzyme conjugates with multiple and single anchoring bonds has been studied under a variety of conditions by radiochemical and enzymological methods. The release of the single‐point‐fixed conjugate could be described by a leakage function, derived previously, yielding the first‐order rate constant of the cleavage of the enzyme‐matrix bond. The nucleophile hydroxylamine increased the detachment rate considerably. The release of the immobilized enzyme was incomplete in all experiments even after prolonged times. The enzyme leakage from multipoint‐attached conjugates was still high enough to prohibit a long‐term use of such preparations in routine work at room temperature.
A New Method for the Purification of RNA Polymerase II (or B) from the Lower Eukaryote Physarum polycephalum
Authors: SMITH Steven S. , BRAUN Richard
Published: March 2005

The DNA‐dependent RNA polymerase II or B from the lower eukaryote Physarum polycephalum has been purified to apparent homogeneity by a new method employing poly(ethylene imine) precipitation and elution, and heparin‐Sepharose affinity chromatography. The method is readily scaled up or down and affords a purification of over 5000‐fold with a yield of 35–45%.

The procedure is easy to perform and can be carried out in less than three days even on a large scale. Furthermore, it gives enzyme of higher purity and in at least 10‐fold greater yield than previously published procedures for its purification from this organism. These improvements have allowed the detection of a series of subforms of the enzyme.

The combination of precipitation using poly(ethylene imine) with chromatography on heparin‐Sepharose may prove useful in the preparation of other proteins which interact with nucleic acids.
Conformations of Purine Ribosyl 5′‐Nucleotides Bound to Glycogen Phosphorylase b
Authors: CHACHATY Claude , FORCHIONI Alain , MORANGE Michel , BUC Henri
Published: March 2005

The effects of the phosphate ion, glucose 1‐phosphate and glycogen on the binding of several nucleotides to phosphorylase b have been investigated by 1H and 2H linewidth measurements and by electron spin resonance. A graphical method is proposed to determine the respective contribution of the strong and weak nucleotide sites of the enzyme to the linewidth of H‐8, H‐2 and H‐1′.

The contribution of the strong site to the linewidth is governed by the residence time of nucleotides in the case of AMP. clAMP and IMP ternary complexes with phosphorylase b and HPO42‐ and by the transverse relaxation time in that of the GMP ternary complex. It is shown that in this latter complex the GMP takes an anti conformation instead of a syn conformation in its binary complex with phosphorylase b. This change is related to an enhanced activity of the complex. The reorientation correlation time of binary and ternary complexes estimated from 2H‐8 linewidth is found of the order of 10−7 s in agreement with our previous 1H linewidth measurements.

The dependence of the binding of a nucleotide on the concentrations of substrates is studied by the electron spin resonance of N6‐(2,2,6,6‐tetramethyl‐piperidine‐4‐yl‐1‐oxy)‐adenosine 5′‐monophosphate. The proton relaxation induced by the nitroxide group of this compound indicates a distance of the order of 1–2 nm between the two nucleotide strong sites of phosphorylase b.
Kinetic Studies of Glyceraldehyde‐3‐Phosphate Dehydrogenase from Rabbit Muscle
Authors: MEUNIER Jean‐Claude , DALZIEL Keith
Published: March 2005

Initial rate studies at pH 7.6 with three aldehydes, product inhibition patterns with NADH and dead‐end inhibition with adenosine diphosphoribose show that the kinetic mechanism of glyceraldehyde‐3‐phosphate dehydrogenase from rabbit muscle cannot be ordered, and support an enzyme‐substitution mechanism. Deviations from Michaelis‐Menten behaviour are consistent with negative interactions in the binding of NAD+ and instability of the species E(NAD)3 and E(NAD)4. Inhibition with large concentrations of phosphate and arsenate indicates competition for a binding site for glyceraldehyde 3‐phosphate, and is not found with glyceraldehyde as substrate.
The Amino‐Acid Sequence of Chicken Fast‐Skeletal‐Muscle Troponin I
Authors: WILKINSON J. Michael , GRAND Roger J. A.
Published: March 2005

The amino acid sequence of troponin I from chicken breast and leg muscle has been determined using trypic and CNBr peptides. The protein contains 182 residues and has a molecular weight of 21 136. The N‐terminus is blocked and is probably acetylated. 35 of the residues have acidic and 45 have basic side chains giving an overall net positive charge of 9. The CNBr peptides from both breast and leg troponin I have been shown to be identical and no heterogeneity has been found in the tryptic peptides isolated from a mixture of the two proteins. It may thus be taken that the two proteins are identical. Comparison with the sequence of troponin I from rabbit fast skeletal muscle shows strong homology with 34 differences out of 182 residues. The major difference is in a deletion of three residues, from 152 to 154, in the rabbit protein.
Freeze‐Fracture Appearance and Disposition of Band 3 Protein from the Human Erythrocyte Membrane in Lipid Vesicles
Authors: GERRITSEN Willem J. , VERKLEY Arie J. , ZWAAL Robert F. A. , VAN DEENEN Laurens L. M.
Published: March 2005

Single bilayer lipid vesicles were formed by removal of Triton X‐100 with Bio Beads SM‐2 from a mixture of egg lecithin and a Triton X‐100 extract of human erythrocyte ghosts. Upon freeze‐fracturing, these vesicles showed intramembrane particles, similar to those seen in the erythrocyte membrane. Similar particles were also observed when a partially purified band 3 preparation was used instead of the crude Triton X‐100 extract.

In the reconstituted vesicles an equal distribution of the intramembrane particles between the two fracture faces was observed. This is in contrast to the unequal distribution of the particles in the erythrocyte membrane, which did not seem to be altered by removal of the extrinsic proteins.

From digestion studies with trypsin and chymotrypsin of vesicles, reconstituted from the crude X‐100 extract, it is concluded that band 3 protein in the vesicle bilayer has a similar orientation as in the native membrane.
Interaction between Penicillin and the dd‐Carboxypeptidase of the Unstable l‐Form of Proteus mirabilis, Strain 19
Authors: SCHILF Wolfgang , FRÈRE Philippe , FRÈRE Jean‐Marie , MARTIN Hans Herbert , GHUYSEN Jean‐Marie , ADRIAENS Paul , MEESSCHAERT Boudewijn
Published: March 2005

Binding of penicillin to the dd‐carboxypeptidase of the unstable spheroplast L‐form of Proteus mirabilis results in the rapid formation of a modified enzyme‐inhibitor complex which in turn under goes rapid decay into reactivated enzyme and an antibiotically inactive penicillin degradation product. Major antibiotic metabolites recovered from such interactions were benzylpenicilloic acid and phenoxymethylpenicilloic acid from benzylpencillin and phenoxymethylpenicillin, respectively, suggesting a second enzymic function of the dd‐carboxypeptidase as a penicillinase of low efficiency. Statistical analyses made with the help of a linear regression program show that the enzyme interacts with the substrate UDP‐N‐acetylmuramoyl‐l‐alanyl‐d‐γ‐glutamyl‐(l)‐meso‐2,6‐diaminopimelyl‐(l)‐d‐alanyl‐d‐alanine and either benzylpencillin or carbenicillin in a non‐competitive manner.
Interaction of Adenylosuccinate Synthetase with F‐Actin
Authors: OGAWA Hirofumi , SHIRAKI Hiroshi , MATSUDA Yoshihiro , NAKAGAWA Hachiro
Published: March 2005

Both crude and purified preparations of adenylosuccinate synthetase from muscle were found to combine with, and dissociate from, muscle debris precipitated from a homogenate of the muscle with water. The binding and dissociation depended on ionic strength. Further study showed that the muscle enzyme was adsorbed to F‐actin, but not to G‐actin or myosin. The muscle‐type enzyme from the liver also associated with F‐actin, but the liver‐type enzyme from the liver did not. In the absence of KCl the molar ratio of adenylosuccinate synthetase from skeletal muscle to actin monomer in F‐actin in the complex formed was 1 to 4. From a Scatchard plot the dissociation constant was calculated to be 0.72 μM. The binding was maximal at pH 5.5–7 in 30 mM potassium phosphate buffer. The complex was completely dissociated in the presence of 0.21 M KCl.

The physiological significance of this binding is discussed on the basis of these findings.
Uptake of Proteoglycans and Sulfated Glycosaminoglycans by Cultured Skin Fibroblasts
Authors: TRUPPE Wolfgang , KRESSE Hans
Published: March 2005

Pinocytosis and degradation of [35S]proteoglycans [35S]glycosaminoglycan chains were studied in cultured skin fibroblasts. Proteoglycans were obtained from fibroblast secretions, glycosaminoglycan chains either by β‐elimination of proteoglycans or from secretions of fibroblasts grown in the presence of d‐xylose or P‐nitrophenyl‐β‐d‐xyloside, respectively. The following results were obtained.

The uptake of proteoglycans as well as of glycosaminoglycan chains is dose‐dependent and follows saturation kinetics. The maximal rate of pinocytosis og glycosaminoglycan chains induced by xylose and P‐nitrophenyl‐β‐d‐xyloside is only about 25% of that of proteoglycans as determined on the basis of the sulfate content. On a molar basis the maximal uptake rate for proteoglycans is 10‐fold lower than for free glycosaminoglycan chains, but proteoglycans have a higher affinity than chains for the proposed receptors.

Uptake is followed by degradation. Its relative proportion is inversely related to the amount of endocytosed material.

Unlabeled proteoglycans compete for the uptake of [35S]proteoglycans, but protein‐free proteoglycan chains are inefficient competitors at the doses used.

In case of [35S]glycosaminoglycans induced by xylose and P‐nitrophenyl‐β‐d‐xyloside, competition for uptake occurs by the addition of unlabeled glycosaminoglycan chains and of hybrid chondroitin‐sulfate‐dermatan‐sulfate decasaccharides, but not by the addition of smaller saccharide fragments.
Thermal Regulation of the Fatty Acid Composition of Lipopolysaccharides and Phospholipids of Proteus mirabilis
Authors: ROTTEM Shlomo , MARKOWITZ Ora , RAZIN Shmuel
Published: March 2005

The fatty acid composition of the lipid A moiety of the lipopolysaccharide and phospholipid fractions of Proteus mirabilis changed significantly on varying the growth temperature. A decrease in the growth temperature from 43°C to 15°C resulted in a decrease in the palmitic acid content of the lipopolysaccharide from 19.4% of total fatty acids at 43°C to 1.4% at 15°C, and by the appearance of an unsaturated fatty acid residue, hexadecenoic acid. Changes in the 3‐hydroxymyristic acid content of the lipid A were minimal. The decrease in the growth temperature also resulted in a decrease in the saturated fatty acid content of the phospholipid fraction, which was accompanied by an increase in their fluidity, as measured by the freedom of motion of spin‐labeled fatty acids incorporated into dispersions made of the phospholipids. Nevertheless, the fluidity obtained with membrane phospholipids extracted from the cells grown at various temperatures were essentially the same when fluidity was determined at the growth temperature, supporting the hypothesis that variations in the fatty acid composition of membrane phospholipids serve to produce membranes having a constant fluidity at different temperatures of growth.
Isolation of Phosphoglycerate Kinases by Affinity Chromatography
Authors: KUNTZ Günter W. K. , EBER Stefan , KESSLER Waltraud , KRIETSCH Helgard , KRIETSCH Wolfgang K. G.
Published: March 2005

A variety of Sepharose derivatives containing dl‐O‐phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate‐oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoly‐dihydrazo‐Sepharose.

The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3‐phosphoglycerate and magnesium ions.

A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85–100%) and reproducability. The capacity of the ATP‐ribosyl‐adipoly‐dihydrazo‐Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogenous.

The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.