bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergism between coenzyme and alcohol binding to liver alcohol dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDÉN Bertil , PETTERSSON Gösta
Published: March 2005

Heterotropic cooperativity effects in the binding of alcohols and NAD+ or NADH to liver alcohol dehydrogenase have been examined by equilibrium measurements and stopped‐flow kinetic studies. Equilibrium data are reported for benzyl alcohol, 2‐chloroethanol, 2,2‐dichloroethanol, and trifluoroethanol binding to free enzyme over the pH range 6–10.

Binary‐complex formation between enzyme and alcohols leads to inner‐sphere coordination of the alcohol to catalytic zinc and shows a pH dependence reflecting the ionization states of zinc‐bound water and the zinc‐bound alcohol. The affinity of the binding protonation state of the enzyme for unionized alcohols increases approximately, by a factor of 10 on complex formation between enzyme and NAD+ or NADH.

The rate and kinetic cooperativity with coenzyme binding of the alcohol association step indicates that, enzyme‐bound alcohols participate in hydrogen bonding interactions which affect the rates of alcohol and coenzyme equilibration with the enzyme without providing any pronounced contribution to the net energetics of alcohol binding.

The pKa values determined for alcohol deprotonation at the binary‐complex level are linearly dependent on those of the free alcohols, and can be readily reconciled with the pKa values attributed to ionization of zinc‐bound water. Alcohol coordination to catalytic zinc provides a major contribution to the pKa shift which ensures that the substrate is bound predominantly as an alcoholate ion in the catalytically productive ternary complex at physiological pH. The additional pKa shift contributed by NAD+ binding is less pronounced, but may be of particular mechanistic interest since it increases the acidity of zinc‐bound alcohols relatively to that of zinc‐bound water.
Low pH induces a hydrophobic domain in the tetanus toxin molecule
Authors: BOQUET Patrice , DUFLOT Edith , HAUTTECOEUR Bernard
Published: March 2005

Binding of the non‐ionic detergent [3H]Triton X‐100 by tetanus toxin, by its fragment C and by its a chain has been studied. At pH 4.00 or above, tetanus toxin does not bind Triton X‐100. At pH lower than 4.00, binding of detergent to the toxin occurs. At pH 3.00, a maximum of 100 mol bound/mol of protein is reached only when the detergent concentration exceeds its critical micelle concentration. No measurable amount of Triton X‐100 is bound by the toxin C fragment at pH 3.00. Most of the tetanus toxin α chain precipitates out in Triton X‐100 at pH 3.00. Leakage of K+ from single‐walled asolectin vesicles loaded with potassium was observed with tetanus toxin at pH lower than 4.00. When ganglioside GDlb was present on the asolectin vesicles, release of K+ was obtained with tetanus toxin between pH 4.00 to 5.00. We suggest that, as for diphtheria toxin, entry of tetanus toxin into an acidic compartment of target cells might be required for the expression of its biological activity.
Nucleotide sequence of the respiratory d‐lactate dehydrogenase gene of Escherichia coli
Authors: CAMPBELL Hugh D. , ROGERS Bruce L. , YOUNG Ian G.
Published: March 2005

The structural gene for the respiratory d‐lactate dehydrogenase of Escherichia coli, a membrane‐bound flavoenzyme, has been subcloned from a 7 × 103‐base‐pair chromosomal HindIII fragment containing the gene [Young, I. G., Jaworowski, A., and Poulis, M. (1982) Biochemistry 21, 2092–2095]. The complete nucleotide sequence of the 2340‐base‐pair PstI‐SmaI subclone has been determined on both strands by the dideoxy chain termination method. A single large open reading frame is present in the nucleotide sequence. The reading frame is preceded by a good ribosome binding site and numerous possible promoter sequences, and is followed by a typical rho‐independent termination sequence. The reading frame predicts that the d‐lactate dehydrogenase polypeptide consists of 571 amino acids (including the initiating methionine residue) with Mr= 64613. The protein does not have a low overall polarity, nor does it contain unusually hydrophobic stretches. It appears to contain a short repeat which is homologous with the well characterized l‐lactate dehydrogenases in the vicinity of the ‘essential’ cysteine residue. Apart from this, homology with other proteins of known sequence has not been detected.
The amino acid sequence of sarcoplasmic calcium‐binding protein obtained from sandworm, Perinereis vancaurica tetradentata
Authors: KOBAYASHI Tomoyoshi , TAKASAKI Yumiko , TAKAGI Takashi , KONISHI Kazuhiko
Published: March 2005

The amino acid sequence of sarcoplasmic calcium‐binding protein obtained from sandworm (Perinereis vancaurica tetradentata) has been determined. It is composed of 174 amino acid residues, its N‐terminus is blocked by an acetyl group and the relative molecular mass is calculated to be 19556. From the results of amino acid sequence, it has three EF‐hand‐type calcium‐binding sites. The positions of calcium‐binding sites could be easily predicted by its hydropathy profile. Amino acid sequence homology between sarcoplasmic calcium‐binding protein from different species is between 14–20%, thus it is not such a conservative protein.
Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1
Authors: MOCZ Gabor , SZILAGYI Laszlo , CHEN LU Renne , FABIAN Ferenc , BALINT Miklos , GERGELY John
Published: March 2005

The kinetics of tryptic breakdown of the heavy chain of chymotryptic myosin subfragment 1 (S1) according to the following scheme (where the numbers respresent approximate masses in kDa) are altered at 21°C by divalent cations (Me2+) and by ATP, ADP, adenosine 5′‐[β, γ‐imino]triphosphate or PPi, with or without Me2+. ATP or its analogs slow step 2 and accelerate steps 3 and 4, while Me2+ accelerates step 2. ATP and its analogs decrease the amount of a transient 27‐kDa peptide [Hozumi, T. & Muhlrad, A. (1981) Biochemistry 20, 2945–2950]. We have found direct evidence for the suggestion in this reference that the 27‐kDa peptide is not an obligatory precursor of the 25‐kDa fragment and that ATP or ADP suppresses the formation of the larger N‐terminal fragment rather than accelerates its breakdown. Cross‐linking of sulfhydryl groups located in the 20‐kDa fragment leads to trapping of MgADP in the N‐terminal 25‐kDa peptide [Wells, J. A. & Yount, R. G. (1980) Biochemistry 19, 1711–1717]; this process affects the tryptic fragmentation of S1 similarly to, but less effectively than, nucleotides. Acts‐S1 formation prevents the effect of ATP on fragmentation. At 37°C S1 loses ATPase activity; tryptic digestion proceeds more rapidly and the 50‐kDa and 25‐kDa fragments are degraded to small peptides. Nucleotides protect against the effects of higher temperature by producing conformational changes not only in the 27‐kDa N‐terminal portion (containing the putative nucleotide binding site) of the heavy chain of S1 but also in the 50‐kDa peptide.
Chemical modification of sulfhydryl groups in p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: VAN BERKEL Willem J. H. , WEIJER Wicher J. , MÜLLER Franz , JEKEL Peter A. , BEINTEMA Jaap J.
Published: March 2005

The cysteine residues in p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens were modified with several cysteine reagents. One of the five sulfhydryl groups reacts rapidly and specifically with N‐ethylmaleimide without inactivation of the enzyme. Cysteine‐116 was found to be the reactive cysteine by isolation of a labeled tryptic peptide.

The enzyme is easily inactivated by mercurial compounds. The original activity can be fully restored by treatment of the modified enzyme with sulfhydryl‐containing compounds. The rate of incorporation of mercurial compounds is pH‐independent and is pseudo‐first‐order up to 90–95% loss of activity. The reaction shows saturation kinetics. The substrate p‐hydroxybenzoate protects the enzyme from fast inactivation. The mercurial compounds themselves inhibit the inactivation reaction at concentrations higher than 80 μM. A spin‐labeled derivative of p‐chloromercuribenzoate reacts fairly specifically with only Cys‐152 on use of enzyme prelabeled with N‐ethylmaleimide, in contrast to p‐chloromercuribenzoate which reacts with additional cysteine residues, i.e. Cys‐211 and Cys‐158. From these results it is concluded that modification of Cys‐152 decreases drastically the affinity of the enzyme for the substrate. The results strongly indicate that the substrate binding site and Cys‐152 are interdependent. This observation is not obvious when the three‐dimensional data only are considered. The modified enzyme exhibits a somewhat higher affinity for NADPH than the native enzyme.

Modification of N‐ethylmaleimide‐prelabeled enzyme by p‐chloromercuribenzoate leads to absorbance difference spectra showing maxima at 250 nm, 290 nm and 360 nm. The intensities of the absorbance difference maxima at 290 nm and 360 nm are strongly dependent on the pH value of the solution. The intensities are very low at low pH values and increase with increasing pH values, reaching a maximum at about pH = 9. The ionizing group shows a pK value of about 7.6. The maximal molar difference absorption coefficient at 290 nm is 3200 M‐1 cm‐1 at pH 9, suggesting that tyrosine residues ionize under the conditions of modification of the enzyme.

The results are discussed in the light of the known three‐dimensional structure.
Nature and location of amide‐bound (R)‐3‐acyloxyacyl groups in lipid A of lipopolysaccharides from various gram‐negative bacteria
Authors: WOLLENWEBER Horst‐Werner , SEYDEL Ulrich , LINDNER Buko , LÜDERITZ Otto , RIETSCHEL Ernst Theodor
Published: March 2005

It has previously been demonstrated [Eur. J. Biochem. 124, 191–198 (1982) and 137, 15–22 (1983)] that the lipid A component of Salmonella and Proteus lipopolysaccharides contains amide‐linked (R)‐3‐acyloxyacyl residues. In the present study lipid A of other gram‐negative bacteria was analysed for the presence of amide‐bound 3‐acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested (Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteriodes fragilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide‐linked (R)‐3‐acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A.

The composition of 3‐acyloxyacyl groups differed considerably among different bacteria. As amide‐bound (R)‐3‐hydroxy fatty acids straight chain and isobranched acyl groups with 10–17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3‐hydroxyl group of 3‐hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10–17 carbon atoms as well as (S)‐2‐hydroxy fatty acids with 12 carbon atoms.

In some cases, using laser desorption mass spectrometry, the distribution of 3‐acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated.
Nucleotide sequence of cloned cDNAs coding for preprosecapin, a major product of queen‐bee venom glands
Authors: VLASAK Reinhard , KREIL Günther
Published: March 2005

Total mRNA from queen‐bee venom glands was transcribed into cDNA and cloned into the PstI and ClaI site of pBR322. The nucleotide sequence of two clones (pUM9/24 and pBMC1) with inserts derived from mRNAs for preprosecapin is presented. The two inserts, which are identical in their overlapping regions, have a coding region encompassing 77 triplets. The cloned cDNAs differ in the length of the 3′‐untranslated region, which is about twice as long in clone pUM9/24. The postulated amino acid sequence of preprosecapin starts with a signal peptide containing an estimated 32 residues, followed by a pro part which terminates with a single arginine. The end‐product secapin is located at the COOH end of the precursor. Activation of prosecapin must, therefore, differ from the processing of promelittin synthesized in the same gland.

Cell‐free translation of total venom gland mRNA in the presence of radioactive histidine yields only two major products, the smaller of which is presumably preprosecapin. Edman degradation of total translation products yields peaks of radioactive tyrosine and histidine at the 4th and 10th cycles respectively. This confirms the sequence of preprosecapin deduced from the cDNA clones. The content of secapin in queen‐bee venom must be higher than it is in worker bee venom.
Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion
Authors: PODESTÁ Ernesto J. , SOLANO Angela R. , MOLINA Y VEDIA Luis , PALADINI Alejandro , SÁNCHEZ Mercedes L. , TORRES Héctor N.
Published: March 2005

Electrofusion of rat adrenal and Leydig cells generated hybrids capable of synthesizing simultaneously both testosterone and corticosterone, under stimulation of lutropin or adrenocorticotropin. Evidence was obtained indicating that under such circumstances, heterologous lutropin receptor—adrenal adenylate cyclase complexes were formed.
Quantification of the different ribosomal phases during the translational elongation cycle in rabbit reticulocyte lysates
Authors: NYGÅRD Odd , NILSSON Lars
Published: March 2005

The proportionality of different ribosomal phases during elongation was estimated in the highly effective rabbit reticulocyte lysate system by use of several complementary analytical methods. The stoichiometric amounts of ribosome‐bound elongation factors EF‐1 and EF‐2 were determined as a measure of ribosomal A‐site occupation. The results were correlated with the puromycin reactivity of the P‐site‐located nascent polypeptide chains. Approximately 25% of the ribosomes were associated with EF‐2, indicating that their A‐sites contained peptidyl‐tRNA. About 35% were associated with EF‐1, signifying that their A‐sites were occupied by aminoacyl‐tRNA. The puromycin reactivity of the nascent chains was approximately 40%. From these data it is concluded that 75% of the peptidyl‐tRNAs were located in the P‐sites and that their puromycin reactivity was limited by the availability of ribosomal A‐sites free for puromycin interaction. After guanosine 5′‐[β, γ‐methylene]triphosphate blockage of the translation, the ribosomal content of elongation factors drastically changed. Under these conditions the proportion of EF‐1‐containing ribosomes increased to approximately 50% while EF‐2‐containing ribosomes decreased to 5%. Concomitantly, the puromycin reactivity increased to approximately 45%. In contrast to previous assumptions the experiments support the view that the elongation rate is limited by the availability of ribosomal A‐sites for the selection of mRNA‐cognate aminoacyl‐tRNAs.
β‐d‐Galactoside transport in Escherichia coli
Authors: KÖNIG Bernhard , SANDERMANN Heinrich
Published: March 2005

The β‐d‐galactoside transport protein (y‐gene product) of Escherichia coli, strain T 206, was solubilized in 85–95% yield using the organic solvents hexamethylphosphoric triamide at pH 7.5 or butan‐1‐ol at pH 4.2. The transport protein obtained with the former solvent could be incorporated into a defined lipid/protein aggregate of density 1.12 g/ml, but no β‐d‐galactoside binding was restored. Diacylglycerol kinase regained activity in the same lipid/protein aggregates. In control experiments, liposomes formed from hexamethylphosphoric triamide were found to be active in the valinomycin‐mediated uptake of Rb+ ions.

β‐d‐Galactoside binding (3.6–5.7 nmol/mg protein) as well as diacylglycerol kinase activity [7 nmol min–1 (mg protein)–1] was reconstituted into proteoliposomes from butan‐1‐ol solution by adaptation of a published procedure [Wright, J. K. et al. (1982) Eur. J. Biochem. 124, 545–552]. A microparticulate nature of the butan‐1‐ol‐solubilized transport protein could be excluded by gel permeation chromatography on a newly synthesized matrix, hydroxypropyl‐Sephacryl S‐300.
Phosphorylation in vivo and in vitro of the arginine‐ornithine periplasmic transport protein of Escherichia coli
Authors: CELIS Roberto T. F.
Published: March 2005

The arginine‐ornithine periplasmic binding protein, an essential component of the arginine‐ornithine transport system of Escherichia coli, was isolated in a phosphorylated form and in a non‐phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies. The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with [γ‐32P]ATP, or by incubation in vitro of the purified binding protein with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic‐shock treatment. The two forms of the protein were separated by DEAE‐Sephacel chromatography.

By several different criteria, which included binding studies, analyses of the amino acid composition of the two forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P‐labeled protein corresponds to a phosphorylated form of the arginine‐ornithine‐binding protein. The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid. The dissociation constant of the phosphorylated protein for arginine was 5.0 μM (dissociation constant of the unmodified protein equals 0.1 μM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane. The pH‐stability profile of the phosphoprotein has a ‘U’‐shape characteristic of acyl phosphates. Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein. These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl function of the protein. This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E. coli, and a step of phosphorylation of the arginine‐ornithine‐binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine‐ornithine transport system.
Amide‐proton exchange studies by two‐dimensional correlated 1H NMR in two chemically modified analogs of the basic pancreatic trypsin inhibitor
Authors: WAGNER Gerhard , STASSINOPOULOU Chariclia I. , WÜTHRICH Kurt
Published: March 2005

The backbone amide proton exchange with the solvent was investigated in 2H2O solutions of the basic pancreatic trypsin inhibitor and two chemical modifications thereof, which were obtained by transamination of the N‐terminus and by cleavage of the disulfide bond 14–38, respectively. The three proteins have nearly identical conformations, but the stability with respect to thermal denaturation is markedly different. Exchange rates for a large number of individually assigned amide protons located both in central and peripheral parts of the protein structures were measured by two‐dimensional correlated spectroscopy (COSY). From analysis of the individual proton exchange rates in the three proteins at different temperatures, an interplay of global and local structure fluctuations was characterized, which promote hydrogen exchange in distinct regions of the molecules. The exchange of particular amide protons may be governed by different motional processes at different temperatures. As a general trend, global fluctuations involving breakage of numerous hydrophilic secondary bonds appear to be dominant at higher temperatures, whereas at lower temperatures the influence of local fluctuations in hydrophobic regions of the protein structures is also clearly noticeable.
Cloning and sequence analysis of human genomic DNA encoding γ subunit precursor of muscle acetylcholine receptor
Authors: SHIBAHARA Shigeki , KUBO Tai , PERSKI Hans Joachim , TAKAHASHI Hideo , NODA Masaharu , NUMA Shosaku
Published: March 2005

Human genomic DNA encoding the γ subunit precursor of the skeletal muscle acetylcholine receptor has been cloned by screening a gene library with a calf cDNA probe and has been subjected to nucleotide sequence analysis. Comparison of the nucleotide sequence of the cloned human genomic DNA with that of the calf cDNA has revealed that the protein‐coding sequence of this gene is divided by 11 introns into 12 exons. Evidence is presented to suggest that the human muscle acetylcholine receptor γ and δ subunit genes are juxtaposed. The primary structure of the γ subunit precursor of the human muscle acetylcholine receptor has been deduced from the corresponding gene sequence. This polypeptide is composed of 517 amino acids including a hydrophobic prepeptide of 22 amino acids. The γ subunit of the human muscle acetylcholine receptor, like the α subunit of the same receptor as well as the α, β and γ subunits of its calf counterpart, shares structural features common to all four subunits of the Torpedo electroplax receptor, such as the putative disulphide bridge corresponding to that in the α subunit proposed as being in close proximity to the acetylcholine binding site and the four putative, hydrophobic transmembrane segments M1 – M4. Thus, the human γ subunit molecule apparently exhibits the same transmembrane topology as proposed for the fish receptor subunits. The 12 exons seem to correspond to different structural and functional domains of the γ subunit precursor molecule. Some exons and the protein regions encoded by them are more highly conserved between the mammalian and Torpedo sequences. The pattern of regional homology observed is consistent with the relatively high conservation of the region encompassing the putative disulphide bridge and of the region containing the putative transmembrane segments M1, M2 and M3.
Mechanisms of activation and secretion of a cell‐associated precursor of an exocellular protease of Pseudomonas aeruginosa 34362A
Authors: FECYCZ Irene T. , CAMPBELL James N.
Published: March 2005

An inactive precursor to the active exocellular protease 1 of Pseudomonas aeruginosa is cell‐associated and located primarily in the periplasmic space. We have studied factors that bring about activation of the precursor in vitro in order to shed some light on the process of its activation and secretion in vivo. A variety of diverse procedures were shown to effect irreversible activation. Several mild non‐enzymatic procedures were effective, such as dialysis of an ammonium sulfate precipitate against neutral buffers, gel filtration (Sephadex G‐100), and ion‐exchange chromatography (DEAE‐cellulose). Activation also resulted following treatment with anionic detergents (sodium dodecyl sulfate, N‐lauroyl sarcosine) and deoxycholate. Limited exposure to any of several proteases with different specificities also resulted in activation. The kinetics of detergent‐catalyzed activation reveals a long lag followed by rapid activation, suggesting at least a two‐stage process. The precursor and the mature protease 1 have indistinguishable molecular masses (33 kDa), as measured by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of these proteins purified by immunoabsorbance chromatography under denaturing conditions. Further, both precursor and protease have identical N‐terminal alanine. Our results suggest that it is improbable that activation is the result of proteolytic processing of the precursor itself, but rather that it may involve the removal of a non‐covalently associated inhibitor molecule. Hydrophobic interaction chromatography on octyl‐Sepharose revealed that activation was accompanied by a significant change in the hydrophobicity, pointing to a significant change in the conformation of the precursor and the mature protease.

A mutant has been studied which accumulates activatable precursor in the periplasm but releases no active enzyme into the culture medium, supporting the hypothesis that secretion through the inner and outer membranes proceed by different mechanisms. Comparison of outer membranes of protease‐secreting strains (34 362A and PAKS 1) and a protease‐negative mutant (PAKS 18) which accumulates precursor has shown that there is a change in the outer membrane protein profile in the latter.
Glycosphingolipids in insects
Authors: DENNIS Roger D. , GEYER Rudolf , EGGE Heinz , MENGES Hans , STIRM Stefan , WIEGANDT Herbert
Published: March 2005

The presence of glycosphingolipids in the pupae of the blowfly, Calliphora vicina, was established. The thin layer chromatographic pattern of the total neutral glycolipids revealed the presence of more than 13 components, the major one being ceramide monohexoside. By the use of high performance liquid chromatography, the three simplest components were isolated and their chemical structures determined: Glc(β1–1)Cer, Man(β1–4)‐Glc(β1–1)Cer [with minor component Gal(β1–4)Glc(β1–1)Cer] and GlcNAc(β1–3)Man(β1–4)Glc(β1–1)‐Cer.

The ceramide composition of the parent insect glycosphingolipids is dominated by the 20:0 fatty acid, arachidic acid, and the sphingoid tetradecasphing‐4‐enine.
Direct evidence by carbon‐13 NMR spectroscopy for the erythro configuration of the sphingoid moiety in Gaucher cerebroside and other natural sphingolipids
Authors: SARMIENTOS Francesco , SCHWARZMANN Günter , SANDHOFF Konrad
Published: March 2005

By comparison of carbon‐13 NMR data from Gaucher cerebroside and from other natural sphingolipids with those from synthetic L‐threo‐ceramide, D‐glucosyl‐L‐threo‐ceramide, D‐erythro‐ceramide and D‐glucosyl‐D‐erythro‐ceramide, we have been able to obtain directly, for the first time, unequivocal evidence for the erythro configuration of the sphingosine moiety in natural sphingolipids. Gaucher cerebroside gave the same data as D‐glucosyl‐D‐erythro‐ceramide. In contrast, significant shift differences have been observed for sphingosine C‐3 (1.9 ppm) and C‐4(1.2 ppm) when comparing the spectral data of Gaucher cerebroside with those of D‐glucosyl‐L‐threo‐ceramide.

Similar differences have also been found between the carbon‐13 NMR data of synthetic D‐erythro‐ceramide and those of L‐threo‐ceramide. In addition, we have observed that threo‐sphingosine C‐5 and C‐4 of ceramide and D‐glucosyl‐ceramide resonate near 129.5 ppm and 133.5 ppm, respectively, with a signal separation of about 4 ppm. On the other hand, these signals from the spectra of synthetic D‐erythro‐ceramide and D‐glucosyl‐D‐erythro‐ceramide as well as from those of gangliosides, lactosyl‐ceramide, galactosyl‐ceramide and sphingomyelin are found near 130 ppm and 135 ppm, respectively, thus being apart by about 5 ppm, irrespective of the degree of glycosylation of the ceramide portion. Thus, by carbon‐13 NMR spectroscopy the erythro configuration of the sphingoid base in natural sphingolipids is directly demonstrated.
Synthesis and secretion of hemopexin in primary cultures of rat hepatocyts
Authors: KATZ Norbert R. , GOLDFARB Valentina , LIEM Heng , MULLER‐EBERHARD Ursula
Published: March 2005

Secretion of hemopexin (20% carbohydrate) and its dependence on glycosylation was studied in primary rat hepatocyte cultures in comparison to the secretion of transferrin (5% carbohydrate).

In pulse‐chase experiments with [35S]methionine half of the labeled hemopexin was secreted in 30 min. By contrast, it took approximately 50 min for secretion of half of the transferrin.

Tunicamycin treatment of cultures significantly delayed the secretion of hemopexin but not that of transferrin.

During the pulse period a proiod a prominent intracellular precursor of hemopexin, smaller than the mature protein, was evident.

It is concluded that the extent of glycosylation of a secretory protein is not necessarily a determinant of the transit time required for intracellular processing and secretion. In the case of hemopexin the glycosylation apparently facilitates the secretion although it is not an absolute prerequisite for the exocytosis of this protein.
DNA regions associated with the nuclear matrix of Ehrlich ascites cells expose single‐stranded sites after deproteinization
Authors: PROBST Hans , HERZOG Richard
Published: March 2005

Ehrlich ascites cells were pulse‐labeled with [3H]thymidine and subjected to prolonged labeling with [14C]thymidine. The isolated nuclei were digested with the restriction endonuclease BspRI and then processed to yield a ‘matrix fraction’ and a ‘non‐matrix fraction’. The DNA fragments purified from these fractions and from whole digested nuclei were examined for nitrocellulose‐binding sites before and after digestion with single‐strand‐specific (S1) nuclease. Both, pulse‐labeled and long‐time‐labeled fragments, isolated from the matrix fraction, exhibited a significantly increased content of nitrocellulose‐binding sites. The major portion of these sites were rendered non‐binding by digestion with single‐strand‐specific nuclease and consisted most probably of structures exposing relatively small stretches of non‐base‐paired DNA. The nature of the minor portion of binding sites which was insensitive to single‐strand‐specific nuclease is not clear. Both types of binding sites are possible candidates for mediating the attachment of DNA to the nuclear matrix.
The cellulase of Trichoderma viride
Authors: BELDMAN Gerrit , SEARLE‐VAN LEEUWEN Marjo F. , ROMBOUTS Frank M. , VORAGEN Fons G. J.
Published: March 2005

Six endoglucanases (Endo I; II; III; IV; V; VI), three exoglucanases (Exo I; II; III) and a β‐glucosidase (β‐gluc I) were isolated from a commercial cellulase preparation derived from Trichoderma viride, using gel filtration on Bio‐Gel, anion exchange on DEAE‐Bio‐Gel A, cation exchange on SE‐Sephadex and affinity chromatography on crystalline cellulose. Molecular masses were determined by polyacrylamide gel electrophoresis. One group of endoglucanases (Endo I, Endo II and Endo IV) with Mr of 50000, 45000 and 23500 were more random in their attack on carboxymethylcellulose than another group (Endo III, Endo V and Endo VI) showing Mr of 58000, 57000 and 53000 respectively. Endo III was identified as a new type of endoglucanase with relatively high activity on crystalline cellulose and moderate activity on carboxymethylcellulose. Exo II and Exo III with Mr of 60500 and 62000 respectively showed distinct adsorption affinities on a column of crystalline cellulose and could be eluted by a pH gradient to alkaline regions. These enzymes were cellobiohydrolases as judged by high‐pressure liquid chromatography of the products obtained from incubation with H3PO4‐swollen cellulose. It was concluded that these exoglucanases are primarily active on newly generated chain ends. Exo I was essentially another type of exoglucanase which in the first instance was able to split off a cellobiose molecule from a chain end and then hydrolyse this molecule in a second step to two glucose units. β‐Gluc I was a new type of aryl‐β‐d‐glucosidase which had no activity on cellobiose. The enzyme had a Mr of 76000 and was moderately active on CM‐cellulose, crystalline cellulose and xylan and highly active on p‐nitrophenyl‐β‐d‐glucose and p‐nitrophenyl‐β‐d‐xylose.
Decrease of fatty acid oxidation, ketogenesis and gluconeogenesis in isolated perfused rat liver by phenylalkyl oxirane carboxylate (B 807‐27) due to inhibition of CPT I (EC 2.3.1.21)
Authors: WOLF Horst P. O. , ENGEL Dieter W.
Published: March 2005

Sodium 2‐[5‐(4‐chlorophenyl)‐pentyl]‐oxirane‐2‐carboxylate (B 807‐27 or POCA) inhibits ketogenesis from endogenous and exogenous long‐chain fatty acids and 14CO2 production from [U‐14C]palmitate, but not from [U‐14C]palmitoylcarnitine or octanoate, and inhibits gluconeogenesis from lactate and pyruvate in perfused livers of starved rats.

Inhibition of ketogenesis, 14CO2 production and gluconeogenesis was complete at concentrations of 10 μmol/1 POCA, but onset was more rapid for inhibition of ketogenesis and CO2 production than for gluconeogenesis. Infusion of octanoate abolished inhibition of all three processes.

Experiments with isolated rat liver mitochondria showed that carnitine palmitoyltransferase I (EC 2.3.1.21) is inhibited by POCA‐CoA.

The inhibitory process is dependent on time and concentration, and more pronounced in mitochondria from fed than from fasted rats. Concentrations required for 50% inhibition after 20 min preincubation with POCA‐CoA are 0.02, 0.06 and 0.1 μmol/l in liver mitochondria from fed, 24‐h‐fasted and 48‐h‐fasted rats, respectively. The inhibitor appears to be tightly bound to the enzyme.

The extent of inhibition of carnitine palmitoyltransferase I correlates well with the hypoglycaemic and hypoketonaemic effects of the compounds in fasted rats. We conclude that specific inhibition of the enzyme leads, due to inhibition of long‐chain fatty acid utilization, to depressed ketogenesis and gluconeogenesis and, in consequence, to hypoglycaemic and hypoketonaemia in vivo under gluconeogenic and ketogenic conditions.
Precursors of ricin and Ricinus communis agglutinin
Authors: LORD J. Michael
Published: March 2005

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000–68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000‐Mr from by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post‐translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth‐surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus.

Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.
Effects of denaturation and methylation on the degradation of proteins in cultured hepatoma cells and in reticulocyte cell‐free systems
Authors: KATZNELSON Revital , KULKA Richard G.
Published: March 2005

Radioiodinated, native and denatured bovine serum albumin (albumin) β‐lactoglobulin and cytochrome c were introduced into hepatoma tissue culture cells by erythrocyte‐ghost‐mediated microinjection, and their rates of degradation were compared. Denatured albumin was degraded at 20% of the rate of undenatured albumin, denatured β‐lactoglobulin was degraded three times faster than undenatured β‐lactoglobulin, while denatured and undenatured cytochrome c were degraded at the same rate. Thus, denaturation does not affect the rates of intracellular breakdown of microinjected proteins in a simple predictable way. Exhaustive methylation did not inhibit the degradation of denatured β‐lactoglobulin or albumin, indicating that, like their undenatured counterparts, they are not degraded via the ubiquitin pathway.

In reticulocyte lysates, in the presence of ATP, denatured albumin and β‐lactoglobulin were broken down at slightly slower rates than the parent proteins. Exhaustive methylation of both denatured and undenatured proteins completely abolished their ATP‐dependent breakdown. This inhibition is consistent with the hypothesis that free – NH2 groups are required for the attachment of ubiquitin prior to degradation in this system.

Removal of an ammonium sulfate fraction from reticulocyte lysates produces a proteolytic system markedly different from the whole lysate [Speiser, S. & Etlinger, J. D. (1983) Proc. Natl Acad. Sci. USA 80, 3577–3580]. In this system both denatured and undenatured albumin and β‐lactoglobulin were degraded essentially independently of ATP. Methylation only slightly decreased the breakdown of denatured proteins, suggesting that they are not degraded via the ubiquitin pathway. A possible explanation of these results is that removal of the ammonium sulfate fraction unmasks an ATP‐independent proteolytic system unrelated to the ubiquitin pathway.
Characterisation of a Dictyostelium discoideum DNA fragment coding for a putative tRNAValGUU gene.
Authors: DINGERMANN Theodor , BERTLING Wolf , PISTEL Friedbert , AMON Elfriede
Published: March 2005

A genomic DNA fragment from Dictyostelium discoideum was characterized. This DNA, although 74% d(A + T)‐rich, codes for a putative tRNAValGUU. The tRNAVal gene overlaps at its 5′ half with another RNA polymerase III transcription unit. This RNA polymerase III transcription unit can be folded into a tRNA‐like shape and is comprised of significant amounts of invariant and semi‐invariant nucleotides present in all eukaryotic tRNAs. This unit contains the two promoter blocks defined for RNA polymerase III, which are homologous to recently defined promoter elements to the extent of 76–88% (A block) and 86–93% (B block) respectively [Sharp et al. (1981) Proc. Natl Acad. Sci. USA 78, 6657–6661]. Both of the overlapping class III genes are transcribed in germinal vesicle extracts prepared from Xenopus laevis oocytes as a single transcription unit, resulting in an unusually large product compared to primary transcripts of other tRNA genes. The unit is not transcribed in HeLa extracts but it competes very strongly for transcription factor(s) under the conditions of stable transcription complex formation. Although the whole unit is transcribed, it is believed that only one functional product is formed. Therefore we define the tRNA‐like structure, coded for on this class III transcription unit, as a putative tRNA ‘pseudogene’ meaning that, although it is transcribed by RNA polymerase III, it is not likely to mature to a functional tRNA.
Isolation and characterization of an iron‐containing superoxide dismutase from tomato leaves, Lycopersicon esculentum
Authors: KWIATOWSKI Jan , SAFIANOWSKA Aleksandra , KANIUGA Zbigniew
Published: March 2005

A cyanide‐insensitive superoxide dismutase was purified from tomato leaves (Lycopersicon esculentum, Mill., var. Venture) to apparent homogeneity. The enzyme had twofold higher specific activity (about 4000 standard units) than ferric superoxide dismutases purified from Brassica campestris [Salin, M. L. and Bridges, S. M. (1980) Arch. Biochem. Biophys. 201, 369–374] and Nuphar luteum [Salin, M. L. and Bridges, S. M. (1982) Plant Physiol. 69, 161–165].

The protein had a relative molecular mass of about 42000 and was composed of two equal subunits noncovalently joined. It was negatively charged (pI = 4.6) and contained about 1.45 mol Fe/mol dimer and negligible amounts of Mn, Cu and Zn. Absorption spectrum and sensitivity to NaN3, H2O2 and temperature are also reminiscent of other ferric superoxide dismutases.

Comparison of amino acid composition indicated, however, a closer relationship to the Mn‐containing enzymes rather than to other Fe‐containing superoxide dismutases. Two possible ways of Fe‐containing superoxide dismutase acquisition by vascular plants were suggested.
Perinatal development of the liver in rat and spiny mouse
Authors: LAMERS Wouter H. , MOOREN Piet G. , DE GRAAF André , CHARLES Robert
Published: March 2005

Rat (Rattus norvegicus) and spiny mouse (Acomys cahirinus) are closely related murinoid species that mainly differ in the developmental timing of birth. A comparison between the developmental profiles of some characteristic enzymes of the liver of both species was carried out to elucidate the question to what extent are these enzymic profiles and hence the maturation of the liver related to the timing of birth? It was found that these organotypic enzymes first become detectable at the same developmental stage in both species. Likewise, the weaning phase of the enzymic profiles occurs at the same developmental time point in both species. It is argued that both the first appearance and the weaning increase in enzyme activity levels occur at endogenously programmed timepoints with only superimposed effects of hormones. In contrast, the perinatal phase of the enzymic profile is completely dependent on the developmental timing of birth and therefore appears not to be anchored to a particular developmental timepoint, but rather to be dependent on birth‐associated (hormonal) adaptation. In accordance with this hypothesis it was found that the morphological development of the liver proceeded independent of the timing of birth. Furthermore, the hormonal regulation of the investigated enzymes was found to be the same in both species. Despite the more advanced state of morphological development of the liver in the spiny mouse at birth, it was found that the inducibility of organotypic gene expression by hormones in spiny mouse fetuses was as limited as in rat fetuses. This observation therefore suggests that the intra‐uterine environment is responsible for the limited inducibility of enzymes before birth.
Nucleotide sequences of the cDNA and an intronless pseudogene for human lactate dehydrogenase‐A isozyme
Authors: TSUJIBO Hiroshi , TIANO Howard F. , LI Steven S.‐L.
Published: March 2005

Eight cDNA clones for lactate dehydrogenase‐A isozyme (LDH‐A) were isolated from a human fibroblast cDNA library, characterized, and no sequence heterogeneity was found. Four cDNA clones appear to contain nearly full‐length cDNA inserts and the complete nucleotide sequence of 1710 base pairs consists of the protein‐coding sequence (999 base pairs), the 5′ (97 base pairs) and 3′ (565 base pairs) untranslated regions and poly(dA) tail (49 base pairs). The predicted amino acid sequence of the human LDH‐A polypeptide shows 92% homology (27 differences out of 331 amino acids compared) with that of the pig LDH‐A subunit determined by direct protein sequencing [Kiltz et al. (1977) Hoppe‐Seyler's Z. Physiol. Chem. 358, 123–127]. Human genomic clones containing an LDH‐A pseudogene were isolated and the nucleotide sequence of 1635 base pairs from an intronless pseudogene was determined. The presence of two termination codons, two deletions of three nucleotides each and the replacement of three arginine residues at the active site (nos 98, 105 and 168) by other amino acids renders its coding region incapable of producing a functional LDH‐A protein. A comparison between human LDH‐A cDNA and the pseudogene sequences reveals 12.9% differences (114 transitions, 65 transversions and 36 deletions/insertions). Further, only four out of the 25 dCpdG dinucleotides present in the cDNA sequence remain unchanged, although the sequences possess 87.1% homology.
Conformation of human IgG subclasses in solution
Authors: KILÁR Ferenc , SIMON István , LAKATOS Susan , VONDERVISZT Ferenc , MEDGYESI György A. , ZÁVODSZKY Péter
Published: March 2005

The structure of six human myeloma proteins: IgG1(Bal), IgG2(Klu), IgG3(Bak), IgG3(Het), IgG4(Kov) and IgG4(Pol), was studied in solution using small‐angle X‐ray scattering and hydrodynamic methods. For IgG1(Bal) and IgG3(Het) the experimental data, including radius of gyration (R°g), radii of gyration of the cross‐section (Rq1, Rq2), intrinsic viscosity [η], sedimentation coefficient (S320,w) and molecular mass, were interpreted in terms of structural models based on the Fab and Fc conformations, observed in crystal, by varying the relative positions of the Fab and Fc parts, i.e. their relative angles and distances.

The values R°g= (6.00 ± 0.05) nm, S°20,W= (6.81 ± 0.10) S and [η] = 0.0062 ± 0.0005 cm3/mg obtained for IgG1(Bal) are compatible with a planar model in which the angle between the Fab arms is about 120°. For IgG3(Het) the following data were obtained: R°g= (4.90 ± 0.05) nm, S°20,W= (6.32 ± 0.01) S and [η] = (0.0065 ± 0.0005) cm3/mg. The apparent contradiction between the higher molecular mass and lower R°g and S°20,W values for IgG3(Het) in comparison to IgG1(Bal) can be resolved by proposing a “non‐planar” (tetrahedral) molecular shape, in which the long hinge peptide is in a folded conformation and the two Fab and Fc parts are in a closely packed arrangement. In this model the angle between the two Fab arms is about 90°, in the average position.

The X‐ray scattering and hydrodynamic behaviour of the IgG2 and IgG4 types of antibodies appeared to be similar to IgG1(Bal). The parameters of the two IgG3 proteins are similar while they are different to the others.
ADP‐ribosylation of phosphorylase kinase and block of phosphate incorporation into the enzyme
Authors: TSUCHIYA Mikako , TANIGAWA Yoshinori , USHIROYAMA Takahisa , MATSUURA Ryoji , SHIMOYAMA Makoto
Published: March 2005

Phosphorylase kinase purified from rabbit skeletal muscle was ADP‐ribosylated by hen liver nuclear ADP‐ribosyltransferase. This modification, as was seen in cAMP‐dependent phosphorylation, was observed only in α and β subunits of the phosphorylase kinase and the latter was more rapidly modified. Analysis of the ADP‐ribosylated amino acid residue sequenced in α and β subunits showed that both subunits were modified at the area of the arginine residue. The Km for NAD was 0.10 mM and the pH optimum was 9.0. When the ADP‐ribosylated phosphorylase kinase was phosphorylated by cAMP‐dependent protein kinase, a reduction in phosphate incorporation occurred with increase in the ADP‐ribosylation. ADP‐ribosylation also suppressed autophosphorylation, to a lesser degree than observed with cAMP‐dependent phosphorylation. The ADP‐ribosylation‐dependent reduction of phosphorylation resulted in a suppression of the phosphorylation‐dependent activation of the phosphorylase kinase. These results together with findings of ADP‐ribosyltransferase activity in the rabbit skeletal muscle [Soman, G. et al. (1984) Biochem. Biophys. Res. Commun. 120, 973–980] suggest that ADP‐ribosylation participates in the regulation of the phosphorylase kinase activity through changes in the rate of phosphorylation.
Structural studies on the linkage unit between poly(galactosylglycerol phosphate) and peptidoglycan in cell walls of Bacillus coagulans
Authors: KAYA Shunji , ARAKI Yoshio , ITO Eiji
Published: March 2005

Structural studies were carried out on the linkage units in the teichoic‐acid–glycopeptide complexes isolated from lysozyme digests of the cell walls of Bacillus coagulans AHU 1366. On treatment with 47% hydrogen fluoride, the complexes gave a disaccharide characterized as glucosyl(β1→4)N‐acetylglucosamine together with major fragments, galactosyl(α1→2)glycerol. By means of Smith degradation and partial acid hydrolysis, the teichoic acid chain was shown to be composed of the repeating units, galactosyl(α1→2)glycerol‐3(1)‐phosphate, which were joined by phosphodiester bonds at C‐6 of the galactose residues.

The mild alkaline hydrolysis of teichoic‐acid‐linked glycan fragments yielded teichoic acid chains and disaccharide‐linked glycan fragments, from which the disaccharide, glucosyl(β1→4)N‐acetylglucosamine, was liberated by mild acid hydrolysis, whereas the same disaccharide linked to the teichoic acid chain was obtained by direct heating of the cell walls at pH 2.5. In addition, the presence of specialized glycerol phosphate units in the linkage region was shown by the isolation of tris(glycerol phosphate)‐3‐glucosyl(β1→4)N‐acetylglucosamine from the products of the Smith degradation of the teichoic‐acid–glycopeptide complexes. Thus, it is concluded that the poly(galactosylglycerol phosphate) chain in the cell walls of B. coagulans AHU 1366 is linked to peptidoglycan through a novel linkage unit, bis(glycerol phosphate)‐3‐glucosyl(β1→4)N‐acetylglucosamine.