bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Protein Glycosylation and Selective Cytotoxicity toward Virally Transformed Fibroblasts Caused by B3‐Tunicamycin
Authors: DUKSIN Dan , Seiberg Miri , MAHONEY Walter C.
Published: March 2005

The biological effect of B3‐tunicamycin, the only known homologue of tunicamycin which contains a saturated fatty‐acid side chain, was examined using chick embryo fibroblasts, a mouse fibroblastic line (3T3) and a virally transformed mouse fibroblastic line (SV40–3T3). This homologue inhibited the transfer of N‐acetylglucosamine 1‐phosphate from UDP‐N‐acetylglucosamine to dolichyl phosphate, catalyzed by microsomes from chick liver or from cultured mouse fibroblasts. B3‐tunicamycin also inhibited the incorporation of mannose into glycoproteins synthesized by chick or mouse fibroblasts. Incorporation of the amino acids proline and tyrosine was inhibited by B3‐tunicamycin to a lesser extent than the incorporation of mannose. The mannose incorporation into glycoproteins synthesized by virally transformed cells was inhibited by B3‐tunicamycin to a higher degree than what was achieved in the nontransformed lines or in the chick primary fibroblats. When the activity of B3‐tunicamycin as an inhibitor of protein glycosylation was compared to other homologues of tunicamycin, it was found to be the most active. This homologue caused complete (more than 95%) inhibition of protein glycosylation at a concentration of 50 ng/ml in chick and in mouse fibroblasts and at a concentration of 10 ng/ml in transformed mouse fibroblasts. When the cytotoxic activities of tunicamycin homologues were examined on nontransformed and virally transformed 3T3 cells, it was found that B3‐tunicamycin displayed the highest selective cytotoxicity toward the transformed cells. When transformed fibroblasts (105 cells/plate) were treated with B3‐tunicamycin (100 ng/ml) for 48 h, complete cell death was observed. The viability and the proliferative activity of the nontransformed fibroblast were normal even when treated with concentrations up to 500 ng/ml of B3‐tunicamycin. This suggests that B3‐tunicamycin may be a suitable candidate for studies of tumor growth in animals.
The Role of Amino‐Terminal Alanine in the Control of Conformation and Activity of α‐Chymotrypsin
Authors: SHARMA Satish K. , HOPKINS Thomas R.
Published: March 2005

Novel acetylated derivatives of three different three‐chained chymotrypsins were prepared from bovine chymotrypsinogen A and their catalytic properties and kinetics of denaturation in urea were compared with those of the corresponding non‐acetylated enzymes.

Measurements of the Km (apparent) as a function of pH confirmed earlier findings of Valenzuela and Bender [J. Biol. Chem. 248, 4909–4914 (1973)] that the α‐species of chymotrypsin is much more sensitive to reversible inactivation at high pH compared to its sister three‐chained chymotrypsins. α1 and K‐chymotrypsin.

Similarly, the denaturation rate constants in 8 M urea of α‐chymotrypsin were much more sensitive to high pH than α1 and K‐chymotrypsin. The urea denaturation study showed a transition at about pH 8 to a more urea‐sensitive from of α‐chymotrypsin, whereas α1 and K‐chymotrypsin were relatively insensitive to a change in pH from 6.5 to 10.

When the N‐terminal Ala149 of α‐chymotrypsin was acetylated, thus preventing protonation of the N terminus, the active enzyme derivative displayed the same Km (app) vs pH profile as α1 and K‐chymotrypsin. Urea denaturation studies with this masked derivative also showed that the pH‐dependent transition of native α‐chymotrypsin at pH 8 was eliminated.

These results demonstrate that it is the presence of the protonated Ala149 residue in α‐chymotrypsin that accounts for much of the hypersensitivity of this enzyme species to inactivation and urea denaturation in the pH region 7.5–10.
Properties of an Aldose Reductase from Pig Lens
Authors: BRANLANT Guy
Published: March 2005

Two monomeric NADPH enzymes from pig lens, an aldehyde reductase and and aldose reductase, have been characterized. The aldose reductase is obtained in a pure form. The aldehyde reductase, usually called hexonate dehydrogenase, is the same protein as that was recently isolated from pig liver [Branlant, G. and Biellmann, J. F. (1980) Eur. J. Biochem. 105, 611–621]. The aldose reductase is shown to have a number of properties in common with the aldehyde reductase, namely its physico‐chemical properties, its tendency to be inhibited by quercitine derivatives and its substrate specificity. These two enzymes differ in their immunological properties. Only aldose reductase has a reactive Cys residue, localized in or near the substrate binding site. In contrast to that shown for aldehyde reductase [Branlant, G. et al. (1981) Eur. J. Biochem. 116, 505–512; Branlant, G. (1982) Eur. J. Biochem. 121, 407–411], no anion‐recognition sites are in the substrate binding site of aldose reductase. The fact that also sugars are substrates for aldose reductase support the idea that this enzyme is implicated in the formation of sugar cataract as suggested by Kinoshita, J. H. et al. [J. Am. Med. Ass. 246, 257–261 (1981)]. Pig lens aldose reductase does not show homotropic cooperative effects with respect to either substrate or coenzyme.
The Core Structure of Shigella sonnei Lipopolysaccharide and the Linkage between O‐ Specific Polysaccharide and the Core Region
Authors: GAMIAN Andrzej , ROMANOWSKA Elčbieta
Published: March 2005

The core structures of Shigella sonnei Phase I, phase II and R‐form lipopolysaccharides have been investigated using the following methods: methylation analysis, Smith and enzymic degradations, 1H‐NMR spectroscopy and passive hemolysis inhibition. The complete structures of the cores occurring in Sh. sonnei, as well as the type of linkage between the core and O‐specific chain, are proposed.
Dynamics of the Spin Transition in Camphor‐Bound Ferric Cytochrome P‐450 versus Temperature, Pressure and Viscosity
Authors: MARDEN Michael C. , Hui bon hoa Gaston
Published: March 2005

The kinetics and equilibrium of the low‐spin (LS) to high‐spin (HS) transition in camphor‐bound ferric cytochrome P‐450 have been measured versus temperature, pressure and visocsity at selected pH and KCl concentration. The results cannot be described by a single set of ΔE°, ΔV° and ΔS, since these parameters change with the solvent conditions. A three‐state model can explain a large bulk of the data but it is clear that other substates are involved. The activation energy and volume are practically zero for the high to low spin transition, while the low to high spin transition parameters vary from 20 kJ/mol to 80 kJ/mol for ΔEΨ and from 5 cm3/mol to 80 cm3mol for ΔVΨ. The observed relaxation time could be varied from about 10 ms to more than 1000 s by changing temperature, pressure and viscosity. BothKLHandKHLrate coefficients scale roughly as l/η, indicating that the slow spin change involves protein motions which are influenced by the solvent viscous forces.
Lipoxygenase mRNA in Rabbit Reticulocytes
Authors: THIELE Bernd J. , ANDREE Hartwig , Höhne Marina , RAPOPORT Samuel M.
Published: March 2005

The synthesis of the erythroid lipoxygenase, an enzyme which is of importance for the degradation of mitochondria during the maturation of reticulocytes to erythrocytes, was studied in reticulocytes from bone marrow and in density‐separated fractions from peripheral blood of anemic rabbits.

Lipoxygenase mRNA was enriched to about 75% by digestion of polysomes with protease K, poly(U)‐Sepharose chromatography and repeated sucrose gradient centrifugation. From sucrose gradient centrifugation, electrophoresis and electron microscopy a molecular weight of about 106 was calculated.

Synthesis of lipoxygenase is absent in erythroblasts, in very young reticulocytes obtained from bone marrow, of in the lightest fractions of reticulocytes from the peripheral blood. More mature blood reticulocytes show a considerable synthesis of the enzyme. The induction of the synthesis of the lipoxygenase seems to be initiated when reticulocytes have reached the peripheral blood.

It is shown that lipoxygenase mRNA is present in reticulocytes as a translationally inactive free cytoplasmic messenger ribonucleoprotein (mRNP) particle. After deproteinization isolated mRNA obtained from masked mRNP codes for authentic lipoxygenase in a cell‐free protein‐synthesizing system of reticulocytes.
A Residue Critical for Flavin Binding in Flavocytochrome b2 from Baker's Yeast
Authors: POMPON Denis , LEDERER Florence
Published: March 2005

The reagent 2‐keto‐3‐butynoic acid is the product formed in the reaction between the suicide reagent 2‐hydroxy‐3‐butynoate and a number of flavoproteins. We describe in this paper the inactivation of flavin‐free flavocytochrome b2 by 2‐keto‐3‐butynoate, in a rapid reaction which introduces 0.9 mol reagent for total inactivation. The modification results in loss of affinity for flavin and affects a cysteine residue.

We also describe in this paper a simple enzymatic method for preparing 2‐keto‐3‐butynoate, as well as some properties of the reagent, in particular its stability and susceptibility to nucleophilic attack. We show that at neutral pH it is highly specific for thiol compounds. Some properties of the adduct formed with glutathione are described. These experiments should pave the way for the use of 2‐keto‐3‐butynoate with other proteins.
Purification and Characterization of Esterase 6A, a Trimeric Esterase of the House Mouse (Mus musculus)
Authors: OEHM Hans‐Christian , DE LOOZE Stephen , RONAI Adam , VON DEIMLING Otto
Published: March 2005

Esterase 6A was isolated from mouse lung and purified 440‐fold by ion‐exchange chromatography, inverse ammonium sulphate gradient solubilization, gel filtration and isoelectric focusing. The resultant product was apparently homogenous by the criteria of polyacrylamide gel electrophoresis and immunodiffusion, and consisted of the electrophoretic form 6A3. A single species of subunit was present on sodium dodecyl sulphate gel electrophoresis. The molecular weight of the native protein was found to be about 178000 with a subunit molecular weight of about 60000. The equivalent weight obtained by active‐site titration with diethyl‐p‐nitrophenyl phosphate was approximately 178000 g/mol, indicating a functional asymmetry in the trimer. The enzyme was shown to have a high affinity for 4‐nitrophenyl hexanoate (Michaelis constant Km= 4.4 μmol/l) with a relatively low catalytic efficiency (catalytic constant kcat= 12 s−1). Esterase 6A was immunologically related to esterase 1 and esterase 9, with which it is genetically closely linked. Further properties of the three esterases were compared.
The Role in CaCO3 Crystallization of an Acid Ca2+‐Binding Polysaccharide Associated with Coccoliths of Emiliania huxleyi
Authors: BORMAN Anton H. , de JONG Elisabeth W. , HUIZINGA Marlies , KOK Dik J. , WESTBROEK Peter , BOSCH Leendert
Published: March 2005

Coccoliths of the unicellular marine alga Emiliania huxleyi are formed intracellularly in a specialized vesicle. Closely associated with the CaCO3 crystals of the coccoliths is an acid Ca2+‐binding polysaccharide. The latter is considered to fulfil a regulatory function in CaCO3 crystallization. In this study it is demonstrated that the coccolith polysaccharide is able to inhibit CaCO3 precipitation in vitro. The degree of inhibition is dependent on the nature of the cations bound to the acid groups of the polysaccharide. After substitution of Na+ by Ca2+ ions the polysaccharide is far less effective in inhibiting CaCO3 precipitation.

The coccolith polysaccharide contains two types of acid groups: uronic acids and sulphate esters. Only the uronic acids are responsible for the inhibition of CaCO3 precipitation. Desulphated polysaccharide inhibits precipitation to the same extent as the native molecule whereas the carboxyl‐reduced polysaccharide is unable to block CaCO3 crystallization.

Inhibition of CaCO3 precipitation can be suppressed by the presence of ethanol. Presumably the conformation of the molecule is altered under these conditions.

Alginic acid [poly(mannuronic‐iduronic acid)] and poly(galacturonic acid) also inhibit CaCO3 precipitation. Inhibition of precipitation is effected by the coccolith polysaccharide and alginic acid at comparable concentrations. The concentration of poly(galacturonic acid) needed to obtain the same result is about 20‐times higher. Contrary to the coccolith polysaccharide, both poly(uronic acid)s inhibit CaCO3 precipitation in the absence as well as in the presence of ethanol. These results suggest that conformational changes of the coccolith polysaccharide may play a role in CaCO3 crystallization in vivo.
A Kinetic Study of Pyrophosphate: Fructose‐6‐Phosphate Phosphotransferase from Potato Tubers
Authors: VAN SCHAFTINGEN Emile , LEDERER Béatrice , BARTRONS Ramon , HERS Henri‐Géry
Published: March 2005

Pyrophosphate: fructose‐6‐phosphate phosphotransferase (PPi‐PFK) has been purified 150‐fold from potato tubers and the kinetic properties of the purified enzyme have been investigated both in the forward and the reverse direction. Saturation curves for fructose 6‐phosphate and also for fructose 1,6‐bisphosphate were sigmoidal whereas those for PPi and Pi were hyperbolic. In the presence of fructose 2,6‐bisphosphate, the affinity for fructose 6‐phosphate and for fructose 1,6‐bisphosphate were greatly increased and the kinetics became Michaëlian. The effect of fructose 2,6‐bisphosphate was increased by the presence of fructose 6‐phosphate and decreased by the presence of Pi. Consequently, the Ka for fructose 2,6‐bisphosphate was as low as 5 nM for the forward reaction and reached 150 nM for the reverse reaction. On the basis of these properties, a procedure allowing one to measure fructose 2,6‐bisphosphate in amounts lower than a picomole, is described.
Chinese Hamster Nuclear Proteins
Authors: PETERS Keith E. , OKADA Tadashi A. , COMINGS David E.
Published: March 2005

A comparison, by two‐dimensional gel electrophoresis, of total interphase nuclear, metaphase chromosomal and nuclear matrix proteins from Chinese hamster V‐79 cells was undertaken to examine the distribution of these proteins during mitosis. We have found a number of differences among these populations, although the two‐dimensional gel patterns are generally similar. The most striking observation is that a loose cluster of six interphase nuclear polypeptides, with isoelectric points in urea between 5.7 and 6.7 and molecular masses ranging from 53 to 75 kDa, is greatly enriched in chromosome preparations. Each of these species is prominent also in the nuclear matrix. Preliminary evidence suggests that one of these polypeptides is the intermediate filament protein vimentin. In addition, two major polypeptides of interphase nuclear preparations, a basic 94‐kDa species and an approximately 65‐kDa species, are absent from chromosomes. The latter polypeptide is the nuclear pore‐lamina complex lamin B. Actin is present in all of these fractions, but tubulin has not been observed. hnRNP particle polypeptides are major components of the nuclear matrix, but are markedly reduced in metaphase chromosomes. The intermediate and basic 65–75‐kDa nuclear matrix polypeptides we have previously demonstrated to be major components of rat liver nuclear matrix, are reduced in Chinese hamster matrix preparations and at least one of these species, a minor, basic, 68‐kDa polypeptide, is missing entirely from metaphase chromosomes. These results are discussed in relation to nuclear and chromosome structure and the possibility of contamination of nuclear protein preparations from cultured cell lines with intermediate filaments.
Cell‐Free Translation and Regulation of Candida tropicalis Catalase Messenger RNA
Authors: YAMADA Takao , TANAKA Atsuo , HORIKAWA Saburo , NUMA Shosaku , FUKUI Saburo
Published: March 2005

To gain information on metabolic control and peroxisome biogenesis in Candida tropicalis growing on n‐alkanes, cell‐free translation of catalase (H2O2: H2O2 oxidoreductase, EC 1.11.1.6), a general marker enzyme of peroxisomes, was performed. The level of catalase activity in alkane‐grown cells was approximately 9‐fold and 27‐fold higher than that in ethanol‐grown and glucose‐grown cells, respectively. Immunochemical titration experiments with rabbit antiserum against the purified peroxisomal catalase from alkane‐grown C. tropicalis indicated that the remarkable variation in the enzyme activity level on different carbon sources was ascribable to a corresponding change in the amount of the enzyme protein. When cell‐free translation was carried out with the mRNA‐dependent reticulocyte lysate system, total RNA prepared from alkane‐grown cells was shown to direct the synthesis of catalase subunit in vitro. The identity of the cell‐free translation product was ascertained by the following evidence: (a) the translation product was immuno‐reactive with specific antibody to catalase and competed effectively with the authentic enzyme for immunoprecipitation; (b) it possessed a molecular weight indistinguishable from that of authentic catalase subunit (Mr 54000); (c) its peptide fragments formed by partial digestion with Staphylococcus aureus V8 protease were identical with those from the authentic enzyme. With the use of the cell‐free translation system, it was indicated that the significant change in the amount of catalase protein on different carbon sources nearly paralleled that in the activity of the mRNA encoding the enzyme.
Amino‐Acid Sequence of a Heat‐Stable Enterotoxin Produced by Human Enterotoxigenic Escherichia coli
Authors: AIMOTO Saburo , TAKAO Toshifumi , SHIMONISHI Yasutsugu , HARA Saburo , TAKEDA Tae , TAKEDA Yoshifumi , MIWATANI Toshio
Published: March 2005

A heat‐stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse‐phase high‐performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn‐Ser‐Ser‐Asn‐Tyr‐Cys‐Cys‐Glu‐Leu‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Thr‐Gly‐Cys‐Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heat‐stable enterotoxin, reported by Moseley et al., except for the C‐terminal Tyr residue.
Expression of Vaccinia Virus Early mRNA in Ehrlich Ascites Tumor Cells
Authors: LEMIEUX Réal , VASSEF Ahmad , BEN‐HAMIDA Fakher , BEAUD Georges
Published: March 2005

Translation of cellular and early vaccinia RNA in nuclease‐treated lysates, derived from uninfected and vaccinia‐virus‐infected cells at the early stage, has been investigated. When using limiting amounts of RNA no discrimination of translation was observed in the infected cells lysates; this conclusion was confirmed by sensitive RNA competition experiments for translation in vitro and also when using two different fractionated systems for protein synthesis in vitro. This absence of detectable discrimination in vitro was established both by comparing incorporation of [35S]methionine into proteins and by analysis of the products thus synthesized by sodium dodecylsulfate gel electrophoresis. However, a modification of the translational machinery from vaccinia‐virus‐infected cells did occur since only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia‐virus‐infected cell lysates may result from the synthesis of an early protein involved in translation or from a better recovery of translational factors from the infected cells, as suggested in the accompanying paper.
Expression of Vaccinia Virus Early mRNA in Ehrlich Ascites Tumor Cells
Authors: LEMIEUX Réal , BEAUD Georges
Published: March 2005

A method for preparing detergent cytoskeletons from uninfected or vaccinia‐virus‐infected cells is described. This method resulted in the fractionation of the cytoplasmic compartment into soluble and cytoskeletal fractions. More than 85% of small molecules and tRNA were released from the cytoskeleton and recovered into the soluble fraction. The cytoskeletal fraction contained about 40% of the cytoplasmic proteins and 67% of total cytoplasmic RNA. Similar values were obtained for vaccinia‐virus‐infected cells. In contrast, whereas 85% of the cellular polysomes and poly(A)‐rich RNA remained associated with the cytoskeleton in uninfected cells, more than 40% of vaccinia early mRNA engaged into polysomes was found to be not associated with the cytoskeleton. A similar partition was found when the viral RNA was labeled for 15 min at 5 min and 20 min after the infection or when varying the duration of the pulse. Soluble and cytoskeletal viral polysomes were found to synthesize a similar set of proteins after translation in vitro of the corresponding mRNA. The fate of rapidly labeled cellular poly(A)‐rich RNA upon vaccinia virus infection was followed by a glucosamine/uridine chase procedure, and also that of relatively stable poly(A)‐rich RNA after long‐term labeling and chase. In both cases no release of poly(A)‐rich RNA from the cytoskeleton occurred after vaccinia virus infection. These experiments reveal that cellular mRNA remains associated to the cytoskeleton in EAT cells infected with vaccinia virus (early period) whereas at least 40% of the vaccinia early polysomes are not associated with the cytoskeleton. A model for vaccinia early mRNA metabolism is presented, which may account for the rapid shut‐off of host protein synthesis.
Thermal Denaturation Studies of Acetylated Nucleosomes and Oligonucleosmes
Authors: YAU Peter , THORNE Alan W. , IMAI Brian S. , MATTHEWS Harry R. , BRADBURY E. Morton
Published: March 2005

The thermal melting behaviors of control and acetylated mononucleosomes, dinucleosomes and trinucleosomes have been studied. Along each series of oligonucleosomes, the melting profiles change in a manner consistent with the increasing number of nucleosomes. For the control mononucleosome, the melting profile exhibits a premelting region at about 61–64°C and a major cooperative transition at 75–77°C. The melting profiles of the control dinucleosomes and trinucleosomes show a premelt at 61–62°C (similar to that of the nucleosome core); an intermediate transition at 73–74°C for the dinucleosome and at 76–77°C for the trinucleosome and a major cooperative transition at 79–80°C for the dinucleosome and at 81–82°C for the trinucleosome. The major cooperative transition at the highest melting temperatures in the melting profiles of the mononucleosome, dinucleosome and trinucleosome comes from the melting of the central region of DNA in the nucleosome strongly complexed with the core histones; the premelt region is attributed to two DNA segments per nucleosome which flank this central DNA region and are free or weakly complexed with histones. The origin of the intermediate transition found for the dinucleosomes and trinucleosomes is not fully understood but probably results from the melting of DNA at the entry to and exit from the nucleosome and the linker DNA which are complexed with histones. A very similar pattern of behavior is observed for the acetylated oligonucleosomes. Direct comparison of the melting profiles of acetylated and control mononucleosomes, dinucleosomes and trinucleosomes show that the premelt region is unaffected by histone acetylation whereas the intermediate and major cooperative transitions for the acetylated oligonucleosomes are broader and occur consistently at lower temperatures than for the controls. These differences support proposals that the N‐terminal regions of core histones interact within the nucleosome and on linker DNA.
Metabolism of Prealbumin in Rats and Changes Induced by Acute Inflammation
Authors: DICKSON Phillip W. , HOWLETT Geoffrey J. , SCHREIBER Gerhard
Published: March 2005

Prealbumin was purified from rat plasma by chromatography on Blue Sepharose CL‐6B, followed by chromatography on concanavalin‐A–Sepharose and preparative electrophoresis in polyacrylamide gel. The overall recovery was 20%. The purified prealbumin was homogeneous upon electrophoresis in detergent containing polyacrylamide gel and was used to raise a monospecific anti‐serum in rabbits. During perfusion of rat liver, [14C]‐leucine was incorporated into prealbumin secreted into the perfusion medium, suggesting that the liver was synthesizing prealbumin. The ratio of the rate of incorporation of leucine into prealbumin to that into total protein was 1.5%. 125I‐prealbumin had a half‐life of 29 h in the bloodstream of healthy Buffalo rats on a diet containing 20% protein. Whole body homogenates from healthy rats contained 6.2 mg prealbumin/100 g body weight. The rate of synthesis of prealbumin, calculated from half‐life and total body pool, was 3.6 mg prealbumin × (100 g body weight)–1× day–1. Two days after induction of inflammation by a subcutaneous injection of turpentine, both the concentration of prealbumin in the plasma and the amount of prealbumin in the whole body homogenates decreased to a minimum of about one third of normal. The relative rate of degradation of 125I‐prealbumin did not change during inflammation. The rate of synthesis of prealbumin decreased considerably, or even ceased, during acute inflammation.
On the Enigma of Old Yellow Enzyme's Spectral Properties
Authors: EWEG Jan Kees , MÜLLER Franz , VAN BERKEL Willem J. H.
Published: March 2005

Old yellow enzyme (NADPH oxidoreductase) in the free and complexed state was thoroughly investigated by the following techniques: absorption, circular dichroism, fluorescence/phosphorescence and electron paramagnetic resonance spectroscopy and fluorescence/phosphorescence decay measurements, applied over a wide range of temperature(7–293 K). The data obtained were interpreted by comparison with results from similar measurements on free FMN, existing spectral data on isoalloxazine model systems and theoretical data.

The results clearly demonstrate the inadequacy of a simple phenolate‐FMN donor‐acceptor charge‐transfer complex to explain the phenomena occurring upon the addition of phenols to old yellow enzyme. Instead it was found that the phenolate anion interferes strongly with an existing tight complex between FMN and the apoprotein, probably an H‐bonded structure in which FMN is tautomerized and interacts with an L‐chiral center. This is concluded from a separate electronic transition with an origin at 496 nm, thus far not recognized as such, and the circular dichroism observed.

The emission is dominated by that of free FMN, although protein‐bound FMN seems also to become luminescent in glassy solution at 143 K. A second fluorescence/phosphorescence emission appears upon excitation of both native and complexed old yellow enzyme in the ultraviolet. This emission is quenched by the addition of phenol to the enzyme, shows a large (3000‐cm–1) blue shift on going to a low‐temperature glass and is tentatively assigned to excimers of nucleic acids. Long‐wavelength excitation with a synchronously pumped, mode‐locked Rhodamine 6‐G dye laser revealed a third, extremely weak emission in both native old yellow enzyme and its complexes. It decays with a lifetime of about 3 ns at 143 K.

Electron paramagnetic resonance spectra revealed the presence of a low amount of an unpaired spin in old yellow enzyme. Owing to an unusual relaxational behaviour it could only be observed below 15 K and, again, the signal was measured in both the native enzyme and its complexes. Possible assignment and consequences of this observation are discussed.

In frozen aqueous solutions of the enzyme‐phenolate complex, a phase transition was discovered at which the colour of the complex reverted to that of the native enzyme. Subsequent melting restored the original colour.

The observed phenomena and existing literature data lead to the conclusion that the only model from which no apparent inconsistencies emerge is that of a very complicated network of hydrogen‐bonded structures in the protein. These involve several, partly unknown, chromophores. Phenols interfere with this network, leading to the formation of the long‐wavelength absorption band in old yellow enzyme.
Biosynthesis and Secretion of α1‐Antitrypsin in Primary Cultures of Rat Hepatocytes
Authors: GROSS Volker , GEIGER Thomas , TRAN‐THI Thuy‐Anh , GAUTHIER Francis , HEINRICH Peter C.
Published: March 2005

The biosynthesis and secretion of α1‐antitrypsin was studied in rat hepatocyte primary cultures. After labeling with [35S]methionine an α1‐antitrypsin with an apparent molecular weight of 49000 estimated by sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis was immunoprecipitated from the cell homogenate. This intracellular form of α1‐antitrypsin could be deglycosylated by endoglycosidase H treatment indicating that its oligosaccharide chains were of the high‐mannose type. Pulse‐chase experiments showed that about 30 min after its synthesis the transformation of the 49000‐Mrα1‐antitrypsin to a protein with an apparent molecular weight of 54000 began. Only this 54000‐Mrprotein was secreted by the hepatocytes. The 54000‐Mrα1‐antitrypsin was not sensitive to endoglycosidase H, but sensitive to neuraminidase, and it incorporated [3H]galactose and [3H]fucose indicating that its oligosaccharide chains were of the complex type. In the presence of tunicamycin, which blocks the formation ofN‐asparagine‐linked oligosaccharide chains, an unglycosylated α1‐antitrypsin with an apparent molecular weight of 41000 was found in the cells as well as in the medium. However, tunicamycin decreased the secretion of α1‐antitrypsin by 60–70%, whereas the secretion of albumin remained unaffected. In the presence of colchicine the secretion of both α1‐antitrypsin and albumin was impaired. The results demonstrate the importance of glycosylation for the secretion of α1‐antitrypsin.
cDNA Cloning and Analysis of Chick‐Embryo‐Liver Cytochrome P‐450 mRNA Induced by Porphyrinogenic Drugs
Authors: BROOKER John D. , O'CONNOR Reina
Published: March 2005

Hepatic microsomal cytochrome P‐450 was elevated in 17‐day chick embryos by combined administration of the porphyrinogenic drugs 2‐allyl‐2‐isopropylacetamide and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine. Increased apoprotein levels were the result of de novo protein synthesis; in vitro obtained translation data suggested that cytochrome P‐450 mRNA levels were elevated.

A 1000‐base cDNA sequence for the drug‐induced cytochrome P‐450 mRNA was isolated from a chick embryo cDNA ‘library’ and this was used as a specific probe to investigate drug‐mediated induction of cytochrome P‐450 mRNA.

RNA‐DNA ‘dot’ hybridisation studies demonstrated that drug treatment led to a 3–5‐fold increase in the level of this mRNA and that the mRNA was predominantly associated with membrane‐bound polyribosomes. Treatment of embryos with the drugs individually demonstrated that both of them induced synthesis of the same mRNA.

These studies show directly that treatment of chick embryos with 2‐allyl‐2‐isopropylacetamide or 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine caused increased levels of cytochrome P‐450 mRNA and suggest that this involved increased transcription of the gene.
Structure of Fibronectin and Its Fragments in Electron Microscopy
Authors: PRICE Todd M. , RUDEE M. L. , PIERSCHBACHER Michael , RUOSLAHTI Erkki
Published: March 2005

Human plasma fibronectin and a series of its large proteolytic fragments were analyzed by electron microscopy using tungsten shadowing on carbon and polystyrene films. On carbon, intact fibronectin appeared as a randomly coiled strand, while on polystyrene it appeared as an elongated structure. Two fragments of fibronectin, Mr=205000 and 190000, which lack the NH2‐terminal domain of fibronectin and retain the collagen‐binding, cell‐attachment and heparin‐binding functions, and a Mr= 170000 fragment, which retains the collagen‐binding and cell‐attachment functions, were seen as rods with varying degrees of nodularity while a Mr= 100000 fragment, which only binds to collagen, had two clear‐cut domains.

These results support the existing biochemical evidence that the segregation of the functional activities in the fibronectin molecule is based on distinct structural domains and provides evidence for the existence of an additional structural domain not revealed by biochemical and functional studies.
Inhibition of Insulin Receptor Binding by Phorbol Esters
Authors: THOMOPOULOS Pierre , TESTA Ugo , GOURDIN Marie‐Françoise , HERVY Catherine , TITEUX Monique , VAINCHENKER William
Published: March 2005

Phorbol esters inhibit the binding of insulin to its receptors on U‐937 monocyte‐like and HL‐60 promyelocytic leukemia human cell lines. Within 20–30 min, exposure of these cells to 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) at 37°C results in a 50% reduction of the specific binding of 125I‐insulin. Half‐maximal inhibition occurs at 1 nM TPA. Other tumor‐promoting phorbol esters also inhibit 125I‐insulin binding in a dose‐dependent manner which parallels their known promoting activity in vivo. TPA does not alter the degradation of the hormone nor does it induce any shedding of its receptors in the medium. The effect of phorbol esters is dependent on temperature and cell type. It is less prominent at 22°C than at 37°C. It is reversible within 2 h at 37°C. TPA reduces the binding of insulin predominantly by increasing its dissociation rate. This effect results in an accelerated turnover of the hormone on its receptors.
Induction of Sporulation in Bacillus brevis
Authors: RISTOW Hansjürgen , PAULUS Henry
Published: March 2005

Under conditions of severe nitrogen starvation, brought about by nutritional shift‐down, Bacillus brevis ATCC 8185 was unable to sporulate unless supplemented with the peptide antibiotic tyrocidine. The induction of sporulation was highly specific for tyrocidine and required only very low concentrations of the peptide (5 μM). Tyrocidine‐induced sporulation was accompanied by the typical sporulation‐specific events (e.g. extracellular protéase production and dipicolinate synthesis) as well as the formation of linear gramicidin. The addition of tyrocidine produced acute inhibition of RNA synthesis that was followed by a limited activation of transcription near the time of onset of linear gramicidin synthesis, when the first sporulation‐specific changes were observed. These results provide direct evidence for a role of tyrocidine in sporulation of B. brevis and suggest that the action of the peptide antibiotic may involve the control of transcription. Such a notion is supported by earlier studies on the effects of tyrocidine and linear gramicidin on purified RNA polymerase.
Purification and Chemical Characterisation of Membrane Glycoproteins from Rat Thymocytes and Brain, Recognised by Monoclonal Antibody MRC OX 2
Authors: BARCLAY A. Neil , WARD Harry A.
Published: March 2005

The MRC OX 2 monoclonal antibody recognises antigens present on rat thymocytes, brain, follicular dendritic cells in lymphoid organs, vascular endothelium, some smooth muscle and B‐lymphocytes. The OX 2 antigens recognised by this antibody were purified from brain and thymus, by solubilisation with sodium deoxycholate, affinity chromatography with MRC OX 2 antibody and gel filtration. The purified brain and thymoeyte OX 2 antigens were glycoproteins with apparent M, 41000 and 47000 respectively as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Rabbit antisera raised against the purified antigens were analysed by radioimmunoassay and immunoperoxidase‐staining of tissue sections. The brain and thymocyte OX 2 antigens were antigenically very similar to those on the other tissues. This indicates that the unusual pattern of distribution was not the result of fortuitous cross‐reaction of the MRC OX 2 antibody, as the rabbit sera would be expected to recognise more determinants on the antigen than that recognised by the monoclonal antibody.

The amino acid compositions of brain and thymus OX 2 antigens were very similar but with no distinguishing features. Carbohydrate compositions showed that the OX 2 antigens were highly glycosylated, with brain OX 2 antigen containing 24% and thymocyte OX 2antigen 33% by weight of carbohydrate. Both OX 2 antigens contained carbohydrate residues typical of structures N‐linked to asparagine but lacked galactosamine, indicating the absence of O‐linked structures. Thymocyte OX 2 contained higher levels of galactose and sialic acid but less fucose than brain OX 2. Similar differences had been observed for brain and thymocyte Thy‐1 antigens and were also observed in pooled glycoproteins purified by lentil lectin affinity chromatography from these tissues, reflecting overall differences in the patterns of glycosylation in the two tissues. The OX 2 antigens showed many similarities to Thy‐1 antigens in their odd patterns of distribution, characteristic migration on polyacrylamide gels in sodium dodecyl sulphate, and carbohydrate compositions. It is possible that OX 2 antigens, like Thy‐1 antigens, have homologies with immunoglobulin domains. A possible role for OX 2 antigens in cell interactions necessary for tissue organisation is discussed.
Hemoglobins from Wistar Rat:
Authors: BRUNORI Maurizio , CONDO Saverio G. , BELLELLI Andrea , GIARDINA Bruno
Published: March 2005

The crystallization properties of the hemolysate and of the purified hemoglobin components from Wistar rat have been studied under various experimental conditions. The results suggest the existence of hemoglobins in the crystalline state even within the red blood cells. However the functional properties of rat erythrocytes are identical to those of whole hemolysate in solution.
Characterization of the Binding Domains in the Fragments Cleaved by Cathepsin G from Human Plasma Fibronectin
Authors: VARTIO Tapio
Published: March 2005

Purified plasma fibronectin was digested by the tissue proteinase cathepsin G isolated from human leukocytes. Using an enzyme‐substrate ratio of 1 : 200 (w/w), fibronectin was degraded to relatively stable peptides of Mr= 38000, 33500, 30000, 28500, 26000, 25000, and 15500 within 4‐8 h. These fragments contained distinct binding domains of the molecule as determined by exposing the peptides to Sepharose‐linked gelatin, arginine, spermidine, spermine and heparin. The bound material was analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The gelatin‐binding activity was located according to the kinetic analysis of the digestion to peptides of Mr= 64000, 40000, and 30000 appearing in that order. Previously uncharacterized domains, having affinity for amino‐containing compounds, were detected by allowing them to bind to Sepharoselinked arginine, spermidine or spermine. The final fragments binding to these molecules had Mr= 25000 and 15 500. The gelatin‐binding domain of fibronectin also bound to these ligands. Heparin‐Sepharose bound several of the 5‐h‐digestion peptides, one of which, with Mr= 28 500, also bound to Staphylococcus aureus and is, on the basis of other studies, located at the NH2 terminus of fibronectin [Mosher and Proctor (1980) Science (Wash. DC) 209, 927‐929]. This peptide was rich in intramolecular disulfide bonds as judged by comparison of the electrophoretic migration under nonreducing and reducing conditions. The results show that in fibronectin there are two distinct heparin‐binding domains. One is located near the COOH terminus of the molecule as described earlier [Ruoslahti et al. (1981) J. Biol. Chem. 256, 7277‐7281] and the other is, according to the present results, located NH2‐terminally between the S. aureus and gelatin‐binding regions. The peptides were eluted from the ligands with 100‐200 mM salt concentrations except for the gelatin‐binding fragment which resisted all concentrations tested (up to 3 M NaCI). In these conditions the peptides were bound generally more weakly than intact plasma fibronectin. In isoelectric focusing the gelatin, arginine and polyamine‐binding peptides had PI = 5.6‐6.2. The heparin‐binding peptides had a PI > 9 and were analyzed using two‐dimensional nonequilibrium pH‐gradient reverse electrophoresis. The binding properties of the fragments suggest an asymmetric cleavage of the subunit polypeptides of fibronectin so that the 64 000‐Mr, gelatin and heparin‐binding fragment is generated from one of the chains whereas the 40000‐Mr, gelatin‐binding and the 30000‐Mr, S. aureus and heparin‐binding fragments are derived from the other one.
Solid‐Phase Sequence Analysis of Polypeptides Eluted from Polyacrylamide Gels
Authors: WALKER John E. , AUFFRET Anthony D. , CARNE Alan , GURNETT Anne , HANISCH Peter , HILL Diana , SARASTE Matti
Published: March 2005

An approach to sequencing proteins by the solid‐phase method combined with isolation of proteins and polypeptides by gel electrophoresis is described. Mixtures of proteins or polypeptides resulting from digests are fractionated in the presence of dodecylsulphate in polyacrylamide gels. They are detected with Coomassie blue, eluted, selectively reacted with porous glass derivatives and sequenced in their amino‐terminal regions with the aid of a new microsequencer. Alternatively they can be analysed or digested with enzymes and fingerprinted. It is a relatively rapid method of purifying proteins for sequence analysis which we have used to provide partial protein sequence data to complement DNA sequences. Nine genes, four from the unc operon of Escherichia coli encoding the α, β, γ and ɛ subunits of ATP synthase and five for capsid proteins of bacteriophage lambda, have been identified by this method.
Affinity Labelling of Yeast Phenylalanyl‐tRNA Synthetase with a 37′‐Oxidised tRNAPhe
Authors: RENAUD Michel , FASIOLO Franco , BALTZINGER Mireille , BOULANGER Yves , REMY Pierre
Published: March 2005

Yeast phenylalanyl‐tRNA synthetase was specifically labelled with a 3′‐oxidised tRNAPhe. Stoichiometric inactivation was achieved with the incorporation of 2 mol oxidised tRNAPhe/mol enzyme which corresponds exactly to the stoichiometry of tRNA binding. The labelled peptide has been isolated using a quick chromatographic procedure which can be applied to any covalent complex formed between a tRNA and an aminoacyl‐tRNA synthetase. The isolated peptide (18 amino acids) was found to encompass the unique cysteine sequence of the smaller β subunit of the enzyme.
Structural Dynamics of Erabutoxin b
Authors: INAGAKI Fuyuhiko , MIYAZAWA Tatsuo , MIYAZAWA Tatsuo , TAMIYA Nobuo , WILLIAMS Robert J. P.
Published: March 2005

A detailed examination has been made of tlie 13C NMR relaxation times of the assigned methyl groups of erabutoxin b. Anisotropic rotation was analysed using a restricted diffusion model. The results are compared with a previous study of the 1H NMR relaxation times. There is good agreement on the segments of the molecule which show restricted motion. Our results are compared with those of previous studies on proteins in solution and in crystals and again tlie general agreement is good. We have attempted to interpret the value of the limited motion seen in tlie protein to the reactions of the neurotoxin.
Limited Proteolysis of the 94000‐Dalton Subunit of Panulirus interruptus Hemocyanin the Carbohydrate Attachment Site
Authors: VEREIJKEN Johan M. , SCHWANDER Edwin H. , SOETER Nell M. , BEINTEMA Jaap J.
Published: March 2005

Limited proteolysis of the native monomeric 94‐kDa subunit of Panulirus interruptus hemocyanin by trypsin, plasmin and subtilisin produces an 18‐kDa fragment, a 71‐kDa fragment and a small glycopeptide. In the plasmin digest a 23‐kDa precursor of the 18‐kDa fragment has been observed. Automatic Edman degradations demonstrated that the 18‐kDa fragments have their origin at the N terminus of the 94‐kDa subunit and incubations with carboxypeptidase A showed that the 71‐kDa fragments originate from the C terminus. The glycopeptide is situated in between. The amino acid sequence of the glycopeptide has been determined. Its carbohydrate content accounts for the total carbohydrate of the 94‐kDa subunit.

All three proteases cleave the subunit at two positions within a very restricted area of the polypeptide chain, which indicates the presence of an exposed loop, carrying the carbohydrate chain, in the corresponding part of the native molecule.
Physicochemical Characterization of Detergent‐Solubilized γ‐Aminobutyric Acid and Benzodiazepine Receptor Proteins from Bovine Brain
Authors: STEPHENSON F. Anne , WATKINS Andrew E. , OLSEN Richard W.
Published: March 2005

[3H]Muscimol and [3H]flunitrazepam binding activities have been solubilized from bovine cortex using the ionic detergent sodium deoxycholate. The soluble receptor proteins were shown to bind [3H]muscimol with a dissociation constant, Kd, of 12 nM and a binding capacity (Bmax value) of 1.56 pmol/mg protein; 7gamma;‐amino[3H]‐ butyric acid with a Kd of 50 nM and Bmax of 1.55 pmol/mg protein; and [3H]flunitrazepam with a Kd of 8 nM and a Bmax of 0.8 pmol/mg protein. Gel filtration of the soluble receptor proteins showed that the γ‐amino[3H]‐butyric acid and [3H]flunitrazepam binding activities comigrated with a Stokes radius of 6.8 nm. The two binding activities were also found to comigrate after sedimentation in a sucrose density gradient. The hydrodynamic properties of the assumed protein‐detergent complexes were determined by gel filtration and sedimentation through gradients of sucrose in H2O or 2H2O. Under the conditions employed, the parameters for both the putative γ‐aminobutyric acid and benzodiazepine receptors were : partial specific volume, 0.73 ml g −1; sedimentation coefficient, 12.5 S; molecular weight, 355000; and frictional ratio 1.46. These observations are consistent with the conclusion that the majority of both binding activities solubilized in deoxycholate reside in a single macromolecular complex. However, Triton X‐100 selectively solubilized the benzodiazepine binding activity. This suggests that the two binding activities can be at least partially separated.
Proteolytic Digestion Studies of Chromatin Core‐Histone Structure
Authors: BÖHM Lothar , BRIAND Gilbert , SAUTIERE Pierre , CRANE‐ROBINSON Colyn
Published: March 2005

Trypsin digestion of chicken erythrocyte chromatin results in a well‐defined set of limit peptides derived from the core histones (P1–P5). Two peptides running in sodium dodecyl sulphate gel electrophoresis at the positions of P2 and P3 have been identified as histone H2B residues 21–125 and 24–125. No C‐terminal residues are therefore lost from histone H2B. The N‐terminal sequence removed correlates well with that having low sequence conservation and not with that showing the greatest basicity. The same correlation was reported previously by us for histone H2A [Eur. J. Biochem. 106, 525–530 (1980)].
Amino‐Acid Sequence of an α‐Parvalbumin, PI = 4.88, from Frog Skeletal Muscle
Authors: JAUREGUI‐ADELL Juan , PECHERE Jean‐Franqois , BRIAND Gilbert , RICHET Colette , DEMAILLE Jaques G.
Published: March 2005

The primary structure of the most basic (PI = 4.88) of the two major parvalbumins from frog skeletal muscle (Rana esculenta) has been determined by partial automatic sequencing of the protein which exhibits a free N terminus, and a study of overlapping peptides obtained by cyanogen bromide cleavage and digestion with trypsin, thermolysin and Armillaria mellea protease.

This protein shows the typical structure of an α‐parvalbumin. Comparison of the primary structure of ionbinding loops of α anld β‐parvalbumins does not provide a clear‐cut explanation of their differences in ionbinding properties [Haiech, J., Derancourt, J., Pechere, J. F., and Demaille, J. G. (1979) Biochemistry, 18, 2752‐2758].
Pancreatic Colipase : Crystallographic and Biochemical Aspects
Authors: PIERROT Marcel , ASTIER Jean‐Pierre , ASTIER Martine , CHARLES Maurice , DRENTH Jan
Published: March 2005

A detailed study of the crystallization of hog and horse colipases has been undertaken. Several crystallographic varieties have been obtained and a 0.3‐nm resolution structure determination is actually in progress. The sequence of the A form of horse colipase (one methionine) is given. From spectrophotometric experiments and sequence comparisons, the involvement of the aromatic residue in position 52 in the miceile binding site has been demonstrated.