bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical and Spectral Properties of Putidamonooxin, the Iron‐Containing and Acid‐Labile‐Sulfur‐Containing Monooxygenase of a 4‐Methoxybenzoate O‐Demethylase from Pseudomonas putida
Authors: BERNHARDT Frithjof‐Hans , HEYMANN Eberhard , TRAYLOR Patricia S.
Published: March 2005

Gel chromatography indicates that putidamonooxin has a molecular weight of about 126000. On the other hand, the amino acid composition and the iron‐to‐protein ratio point to a minimal molecular weight of 33000 and 31000 respectively. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme migrated as a homogeneous band corresponding to a molecular weight of about 40000. The number of spots found in the tryptic peptide map of the carboxymethylated and digested enzyme indicates that putidamonooxin is composed of three or four identical subunits. After covalent cross‐linking of the subunits with dimethyl suberimidate and subsequent dodecylsulfate electrophoresis the main bands were in the molecular weight range of 40000, 87000 and 124000. These findings lead us to propose that putidamonooxin is either a trimer or tetramer.

The amino acid composition of putidamonooxin and related data calculated from this are given. The isoelectric point was shown by isoelectric focusing to be at pH 4.7. Low‐temperature optical spectra of the reduced and oxidized enzyme as well as of three different putidamonooxin substrate complexes are given together with those recorded at 10°C. Enzyme · substrate binding spectra are observed with the oxidized putidamonooxin but not with the reduced enzyme. For the oxidized putidamonooxin a molar absorption coefficient at 455 nm of 14.7 mM−1 cm−1 was determined. Ks values of putidamonooxin towards different substrates and substrate analogues (i.e. tight couplers, partial uncouplers and uncouplers) are presented and possible reasons for the difference between the Ks values here obtained and the previously reported Km values are discussed.
The 50‐S Ribosomal Subunit Inhibits Functional Interaction between the 30‐S Subunit and Phage RNA
Authors: ZIPORI Pnina , BOSCH Leendert , VAN DIEIJEN Gerbrand , VAN DER HOFSTAD Gerard A. J. M.
Published: March 2005

Vacant 30‐S subunits treated with the 7‐S immunoglobulin G fraction of sera from rabbits, immunized against ribosomal protein S1, (anti‐S1) lose their capacity to bind phage RNA. However, upon interaction of the subunits with MS2 phage RNA at 37°C, complexes between the 30‐S subunits and MS2 RNA (30‐S · MS2‐RNA) are obtained, which do not dissociate under the effect of anti‐S1.

The capacity of such complexes to carry out protein synthesis in the presence of anti‐S1 has been examined. It is found that only a 30‐S · MS2‐RNA complex which is formed in the presence of initiation factor IF‐3 and in the absence of 50‐S subunits is resistant to anti‐S1 throughout the translation process. In contrast, when a complex formed under similar conditions but in the presence of 50‐S subunits is tested in this way, it is found to be sensitive to anti‐S1.

The inhibition derived from 50‐S subunits is relieved if IF‐2 and tRNAMetf are present, in addition to IF‐3, during the formation of the ribosome · mRNA complex.

It is concluded that the 50‐S subunit suppresses the potential of the 30‐S subunit to attach properly to phage RNA and induces the formation of an immature 30‐S · MS2‐RNA complex. Possible mechanisms by which the 50‐S particle inhibits the formation of the mature 30‐S · MS2‐RNA complex are discussed.

The data are consistent with the view that displacement of the 50‐S ribosomal subunit is essential for initiation of protein synthesis on natural messengers.
N‐(2‐Oxoacyl)amino Acids and Nitriles as Final Products of Dipeptide Chlorination Mediated by the Myeloperoxidase/H2O2/Cl− System
Authors: STELMASZYŃSKA Teresa , ZGLICZYNSKI Jan M.
Published: March 2005

The chlorination of dipeptides by the myeloperoxidase/H2O2/Cl− system takes place at the N‐terminal amino group, whereas no chlorination of the amide nitrogen of the peptide bond can be observed. The N‐terminal amino group is chlorinated to N‐monochloroamine or/and N‐dichloroamine. N‐Monochloropeptides were the main products at higher pH values, at lower pH a mixture of N‐monochloropeptides andN‐dichloropeptides was formed owing to the dismutation of N‐monochloroamine to N‐dichloroamine.

N‐Monochloropeptides decompose, yielding NH3 and the corresponding N‐(2‐oxoacyl)amino acids. N‐Dichlorodipeptides decompose faster but to nitriles and the free C‐terminal amino acids. N‐Dichloroglycyl‐amino acid decomposes through a relatively stable intermediate (cyano‐formylamino acid) to hydrogen cyanide, cyanogen chloride and the free C‐terminal amino acid.

Insulin chlorination also yields N‐terminal glycyl and phenylalanyl N‐monochloro derivatives, which deaminate to glyoxylyl and phenylpyruvyl residues.
A Systematic Study of Effects of Non‐Ionic Detergents on Solubilization and Activity of Pig Kidney Adenylate Cyclase
Authors: GUILLON Gilles , ROY Christian , JARD Serge
Published: March 2005

The effects of 17 non‐ionic detergents on pig kidney plasma membrane adenylate cyclase were tested (digitonin; detergents from the Triton series: X45, X100, X102, X114, X165, X305, X405, N101; the Brij series: 35, 56, 96 and the Tween series: 20, 40, 60, 80, 85). Membranes (2.66 mg protein/ml) were treated with increasing amounts of detergent for 150 min at 0°C. The adenylate cyclase activities present in treated membranes and in their corresponding non‐sedimentable fractions were measured at 30°C. Only detergents having hydrophilic lipophilic balance values from 12 to 14 were effective solubilizing agents. For these detergents, the solubilization yield was dependent on their structure. When solubilized adenylate cyclase kept at 0°C was incubated at 30°C, the enzyme catalytic activity exponentially decreased towards an equilibrium value with a half‐time of 5 min. This temperature‐dependent adjustment of enzyme activity was reversible. Its magnitude was dependent on the detergent used. A more progressive but less pronounced effect was observed with non‐solubilizing detergents. The micellar form of detergent in the incubation medium was found to be responsible for the appearance of microheterogeneity in the population of solubilized enzyme molecules differing by the substrate accessibility. This effect was suppressed when reducing the detergent concentration in the incubation medium.
Incorporation of 6‐Thioguanosine and 4‐Thiouridine into RNA
Authors: MELVIN William T. , MILNE Helen B. , SLATER Alison A. , ALLEN Hamish J. , KEIR Hamish M.
Published: March 2005

Isolation of newly synthesised RNA can be achieved by treatment of cells in culture with 6‐thioguanosine or 4‐thiouridine followed by separation of thiol‐containing RNA by affinity chromatography on mercurated cellulose columns. After short periods of treatment with 6‐thioguanosine the proportion of RNA retained on mercurated cellulose is the same for both poly(A)‐containing and poly(A)‐free RNA, indicating similar incorporation of the drug into mRNA and rRNA. However, after longer periods of exposure, the cytotoxic effect of 6‐thioguanosine results in diminished incorporation of radioactive uridine into RNA and of radioactive leucine into protein; this suggests that synthesis of both RNA and protein are impaired. On the other hand, even after long exposure to high concentrations of 4‐thioridine, the syntheses of RNA and protein are not significantly affected. Proteins synthesised after treatment of cells with 6‐thioguanosine are less stable than proteins synthesised after treatment of cells with 4‐thiouridine.
Effect of Various Nucleotides on Folding and Enzymic Properties of a Synthetic 63‐Residue Analog of Ribonuclease A and Natural Ribonuclease A
Authors: GUTTE Bernd
Published: March 2005

When a reduced synthetic 63‐residue analog of ribonuclease A (RNase A) was reoxidized in the presence of 2′‐CMP, a competitive inhibitor of RNase A, the overall activity after removal of the inhibitor was lower than that of the same analog reoxidized in the absence of 2′‐CMP. However, reoxidation in the presence of 2′‐CMP diminished the activity toward poly(U) more strongly than that toward poly(C).

To investigate the influence of substrate analogs on folding and enzymic properties of the natural enzyme, reduced RNase A was reoxidized in the presence of 3′‐CMP, 3′‐UMP, adenosine 2′:3′‐monophosphate (2′:3′‐AMP) or guanosine 2′:3′‐monophosphate (2′:3′‐GMP). The activities of ribonuclease reoxidized in the presence of 3′‐CMP were essentially unaltered whereas RNase A refolded in the presence of 3′‐UMP had 150% activity toward 2′:3′‐UMP and RNase A refolded in the presence of 2′:3′‐AMP had 170% activity toward poly(A) as compared with the activity of untreated natural RNase A against these substrates.

Kinetic studies using uridine 2′:3′‐monophosphate (2′:3′‐UMP) as substrate showed that the maximum velocity of the reaction catalyzed by RNase A which had been reoxidized in the presence of 3′‐UMP was twice that found with untreated native RNase A as enzyme. Comparison of the Michaelis constants indicated that refolding of RNase A in the presence of 3′‐UMP did not increase the affinity for 2′:3′‐UMP.

The conformational isomers of RNase A formed through reoxidation in the presence of various nucleotides were not very stable. Thus, raising the temperature to 40°C lowered the activity against poly(A) from 170% to 115%. However, it cannot be excluded that conformational isomers of this kind are also formed during the biosynthesis of RNase A.
Purification and Properties of a New Restriction Endonuclease from Haemophilus influenzae Rf
Authors: KAUC Leszek , PIEKAROWICZ Andrzej
Published: March 2005

Haemophilus influenzae Rf 232, showing the phenomena of restriction and modification, contains an endonuclease that inactivates in vitro the biological activity of DNAs lacking the strain‐specific modification. This specific restriction endonuclease has been purified to near homogeneity by a procedure that includes DNA‐agarose chromatography. This highly purified enzyme requires ATP and Mg2+ for activity and is stimulated by S‐adenosylmethionine. The enzyme seems to cleave DNA at well‐defined sites, since it produces a specific pattern of bands upon agarose gel electrophoresis. The enzyme has no ATPase activity. A methylase activity is observed in the course of the endonucleolytic reaction, which probably protects some of the DNA sites from cleavage.
FAD Is Covalently Attached to Peptidyl‐tRNA during Cell‐free Synthesis of 6‐Hydroxy‐d‐nicotine Oxidase
Authors: HAMM Hans‐Heinrich , DECKER Karl
Published: March 2005

The process, by which FAD is attached covalently to the 6‐hydroxy‐d‐nicotine oxidase apoprotein in d‐nicotine‐induced cells of Arthrobacter oxidans was studied in vitro. [3H]Adenine‐labelled FAD prepared biosynthetically in Clostridium kluyveri was incorporated into the 6‐hydroxy‐d‐nicotine oxidase molecule during cell‐free translation. FAD rather than FMN or riboflavin was thus shown to be the flavin derivative transferred to the polypeptide chain. After short‐term protein synthesis on ribosomes from induced A. oxidans cells in the presence of an Escherichia coli MRE 600 supernatant fraction and [adenine‐2‐3H]FAD, the peptidyl‐tRNA fraction was separated from completed polypeptides. Labelled FAD was found to be covalently attached to the tRNA‐bound polypeptides. Cleavage of the tRNA‐peptide bond released labelled polypeptides the largest of which migrated as authentic 6‐hydroxy‐d‐nicotine oxidase during dodecylsulfate/polyacrylamide gel electrophoresis. These results strongly suggest that FAD is incroporated into the nascent polypeptide chains of 6‐hydroxy‐d‐nicotine oxidase during ribosomal translation.
Protons and Mg2+ Cations as Probes in Investigating the Role of GTP in Initiation Complex Formation
Authors: BEAUDRY Patrice , BEGARD Evelyne , Pierre DOUZOU  , GRUNBERG‐MANAGO Marianne
Published: March 2005

fMet‐tRNAfMet binding to both 30‐S subunits and to 70‐S particles is dependent on both pH and Mg2+ concentration: for fMet‐tRNAfMet binding to 70‐S particles, variations of pH and Mg2+ concentration are tightly interdependent. This behavior can be interpreted by the polyelectrolyte theory as a direct consequence of the fact that the binding occurs in a polyanionic microenvironment. The pH‐dependent binding to 70‐S particles clearly shows the involvement of two prototropic groups which appear to be those carrying out GTP hydrolysis, therefore directly linked to initiation complex formation; in the presence of a non‐hydrolyzable analogue to GTP, guanosine 5′‐[β,γ‐imido]triphosphate, the binding of fMet‐tRNAfMet shows much less interdependence between variation of pH and Mg2+ concentration.
Bacteriophages of Streptococcus pneumoniae
Authors: GARCÍA Ernesto , RONDA Concepción , LÓPEZ Rubens
Published: March 2005

The properties of Dp‐4 phage and its DNA have been studied. The phage has a polyhedral head of 60 nm diameter, a tail 155 nm long and a buoyant density in CsCl of 1.48 g/cm3. Analysis by acrylamide gel electrophoresis indicates the presence of five polypeptides. Dp‐4 DNA has a molecular weight of 37 * 106, a melting point of 83.5°C (when dissolved in 0.15 M NaCl/0.015 M sodium citrate, pH 7.0) and a G + C content of about 33%. Denaturation of DNA yields two strands of different buoyant density in a neutral CsCl gradient. The interaction of poly(U,G) with the heavy strand strongly enhances its density and allows the preparative separation of both strands. Native Dp‐4 DNA has unusual physical properties: abnormally low buoyant densities both in CsCl (1.666 g/cm3) and in Cs2SO4 (1.410 g/cm3) (which do not correspond to the value predictable from the G + C content of the DNA), and a high thermal stability at low ionic strength.
Ascorbate Oxidase from Cucurbita pepo medullosa
Authors: MARCHESINI Augusto , KRONECK Peter M. H.
Published: March 2005

Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low‐molecular‐weight copper protein containing one type‐1 copper/20000 Mr could be separated during the purification of the oxidase. The six‐step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 ± 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 ± 50 units/mg protein or 1088 ± 15 units/μ copper.

The pure enzyme is characterized by the following optical purity indices: A280/A610= 25 ± 0.5, A330/A610= 0.65 ± 0.05 and A610/A500= 7.0 ± 0.25. The molar absorption coefficient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts to 9700 M−1 cm−1.

Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type‐1 (blue) copper gz= 2.227, gy= 2.058, gx= 2.036; Az= 5.0 mT, Ay=Ax= 0.5 mT, for the type‐2 (non‐blue) copper g‖= 2.242, g⊥, = 2.053 A‖= 19.0mT, A⊥= 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type‐1 class, and one to the type‐2 class, as demonstrated by computer simulations of the EPR spectra.

To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L‐ascorbate or reductate must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (λexmax= 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type‐3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310nm(Δɛ= 1000 ± 50 M−1 em−1). Corresponding changes in EPR and fluorescence spectra have not been detected.
Transfer of the 1‐pro‐R and the 1‐pro‐S Hydrogen Atoms of Ethanol into Krebs‐Cycle and Related Acids in vivo
Authors: BLOMBERG Sverker , CRONHOLM Tomas
Published: March 2005

A method for gas chromatographic/mass spectrometric analysis of low‐molecular‐weight organic acids in liver tissue is described. The method involves formation of methoximes of oxo acids, preventing exchange of hydrogen by enolization. The acids are separated as methyl esters by gas chromatography on an SP‐1000 capillary column. The origin of fragment ions in the mass spectrometric analysis was studied by analysis of deuterated analogs.

Lactate, pyruvate, 3‐hydroxybutyrate, fumarate, succinate, malate, 2‐oxoglutarate and citrate were analyzed in freeze‐clamped livers of rats given [1, 1‐2H]ethanol, (1R)‐[1‐2H]ethanol and (1S)‐[1‐2H]ethanol. No deuterium was incorporated in succinate and the oxo acids analyzed. The incorporation of deuterium from [1, 1‐2H2]ethanol in one position of malate and fumarate was about the same as in lactate (18–23% of the atoms), but the deuterium in lactate originated to a larger extent from the 1‐pro‐R position than in malate and fumarate. This indicates lack of equilibration of the NADH pools utilized by cytosolic malate dehydrogenase and lactate dehydrogenase, and that the former pool is more closely related to cytosolic acetaldehyde oxidation. 3‐Hydroxybutyrate incorporated the 1‐pro‐S hydrogen of ethanol to a larger extent than the 1‐pro‐R hydrogen. This is in agreement with the presence of 3‐hydroxybutyrate dehydrogenase and aldehyde dehydrogenase in the mitochondrial compartment. Hydrogen in citrate appears to be derived from C‐1 of ethanol both via oxaloacetate and via intramitochondrial isocitrate dehydrogenase.
Desialized Low‐Density Lipoprotein Regulates Cholesterol Metabolism in Receptor‐Deficient Fibroblasts
Authors: FILIPOVIC Ivan , BUDDECKE Eckhart
Published: March 2005

Binding of homologous low density lipoproteins (LDL) to specific cell‐membrane receptors initiates regulation of cholesterol metabolism in normal human fibroblasts, but minor regulatory effects are observed in receptor‐deficient cells. After enzymic removal of sialic acid residues, however, the desialized LDL aquires the ability to regulate cholesterol biosynthesis and cholesterol esterification in receptor‐deficient cells as effective as native LDL in normal cells. This is indicated by a suppression of hydroxymethylglutaryl‐CoA reductase activity, by a reduced incorporation of [14C]acetate into cholesterol and by a stimulation of the [3H]oleate incorporation into cholesterol esters in both normal and receptor deficient cells. Competitive binding and internalisation experiments revealed that the action of desialized LDL in normal and receptor‐deficient cells is receptor‐specific.

The results suggest that desialisation of LDL facilitates its binding to cell membrane receptors, its internalisation and subsequent regulation of cellular cholesterol metabolism, this effect becoming significant in fibroblasts with reduced number of LDL receptors.
Translation by Escherichia coli Ribosomes of Alfalfa Mosaic Virus RNA 4 Can Be Initiated at Two Sites on the Monocistronic Message
Authors: CASTEL Ad , KRAAL Barend , KONIECZNY Andrzej , BOSCH Leendert
Published: March 2005

Substantial evidence is provided to corroborate our previous finding that Escherichia coli ribosomes recognize two binding sites on the 5′ end of alfalfa mosaic virus (AMV) RNA 4 [for a preliminary report see Castel, A., Kraal, B., Kerklaan, P. R. M., Klok, J., and Bosch, L. (1977) Proc. Natl Acad. Sci. U. S. A. 74, 5509–5513]. Translation can start at either site using AcPhe‐tRNA or fMet‐tRNA as initiator and takes place in the same reading frame along the monocistronic mRNA. The size and composition of the isolated extra NH2‐terminal fragment of the acetylphenylalanyl product were found to be in agreement with the 5′ non‐coding region of the messenger. Removal of the 5′‐terminal cap structure of AMV RNA 4 did not influence significantly both initiation reactions. Ribosomal protein S1 was essential for binding as well as incorporation of both fMet‐tRNA and AcPhe‐tRNA. A similar interaction on the ribosome was found for AcPhe‐tRNA directed by AMV RNA 4 as for fMet‐tRNA directed by either AMV RNA 4 or MS2 RNA with respect to the influence of initiation factors. It is concluded that the heterologous plant viral messenger is reliably translated in the E. coli system and that E. coli ribosomes recognize with high specificity an extra initiation site close to the 5′ extremity of the messenger. The relationship of this site to a hypothetical entry site involved in the early recognition in the initiation mechanism between ribosome and messenger is discussed.
Thermostable Endogenous Inhibitors of Cathepsins B and H
Authors: LENNEY James F. , TOLAN Jeffrey R. , SUGAI Wesley J. , LEE Alan G.
Published: March 2005

Separate thermostable inhibitors of cathepsins B and H (IB and IH) were found in every rat and human tissue that was analyzed. The inhibitors were separated from the cathepsins by heating at 80–90°C at pH 3. IB and IH from rat lung were purified and found to occur in multiple molecular forms. Rat lung IB and IH were very similar proteins with molecular weights of 14000. IB and IH from hog kidney were also purified and IH was obtained free of IB. These inhibitors also displayed microheterogeneity and had molecular weights of 11000. Hog kidney IH inhibited chymopapain, but did not inhibit bromelain, chymotrypsin or cathepsin D. Rat and human serum contained thermostable inhibitors of cathepsins B and H, but these molecules had relatively high molecular weights. Therefore the low‐molecular‐weight IB and IH from other tissues appear to be intracellular in origin. When extracts of kidney or liver were autolyzed at pH 3–6, there was a large increase in cathepsin B or H activity with a concomitant decrease in IB and IH. Inhibitor destruction was apparently catalyzed by a proteinase other than cathepsin D or E. Rat kidney, liver, and spleen were rich sources of cathepsins B and H, the kidney yielding three times as much activity per gram as liver or spleen. Hog kidney was rich in cathepsin H, but yielded little or no cathepsin B. IB and IH were also present in protozoa, luna fish, chicken and toad. The inhibitors from Tetrahymena pyriformis inhibited the thiol proteinase from this protozoan.
Exchange of ADP, ATP and 1:N6‐Ethenoadenosine 5′‐Triphosphate at G‐Actin
Authors: ENGEL Jürgen , NEIDL Cornelia
Published: March 2005

Nucleotide exchange was monitored by the strong increase of fluorescence accompanying binding of 1:N6‐ethanoadenosine 5′‐triphosphate (ɛATP) to G‐actin. ɛATP · G‐actin was titrated by ADP and a relative binding constant KADP/ɛATP= 0.03 was obtained. With KɛATP/ATP= 0.19, the physiologically important relative binding constant KADP/ATP= 0.0057 was calculated. From kinetic measurements of the exchange reaction a rate constant of ADP dissociation k−= 8 × 10−4 s−1 was derived which was compared with the corresponding values for ATP (8 × 10−4 S−1) and eATP (4 × 10−3 S−1) (all values in 20 mM Tris · HC1 buffer, pH 8.2 containing 0.8 mM CaCl2 at21°C).

The exchange of ADP versus ATP is an important step in the steady‐state equilibrium of actin polymerization. Since polymerization of ATP · G‐actin to ADP · F‐actin is irreversible, depolymerization can take place only by dissociation of ADP · G‐actin followed by nucleotide exchange. It was found from a comparison of k‐ with the rate constants of the polymerization steps that the ratio of ADP · G‐actin to ATP · G‐actin is much higher in the steady state than at equilibrium.
Metabolic Carbohydrate Labelling of Glycoproteins from Mitogen‐Stimulated Mouse Lymphocytes
Authors: VAN EIJK Ronald V. W. , ROSENFELDER Günter , MÜHLRADT Peter F.
Published: March 2005

CBA/J mouse lymphocytes from thymus, spleen and lymph nodes were separated into fractions enriched in either T (thymus‐derived) or B (bone‐marrow‐derived) cells. The cells were stimulated with ‘T cell mitogens’ (concanavalin A or phytohemagglutinin), ‘B cell mitogens’ (lipopolysaccharide or lipoprotein), and pokeweed mitogen to incorporate tritiated mannose, galactose or fucose. Mitogen‐induced incorporation of carbohydrates into acid‐precipitable material proved to be specific for the cell population.

Carbohydrate‐labelled cells were extracted with dodecyl sulfate buffer and the extracts subjected to polyacrylamide gel electrophoresis and subsequent autoradiography. Depending on the carbohydrate used for labelling several glycoproteins specific for activated T cells (GP 200, GP 170, GP 125) and activated B cells (GP 210, GP 85 and GP 75) were noted. Also T or B cell subpopulations could be distinguished by their glycoprotein patterns. From these patterns the following conclusions were derived.

a) Concanavalin‐A‐reactive T cells from thymus, spleen and lymph nodes comprise different subpopulations.

b) Concanavalin A and phytohemagglutinin stimulate the same T cell subpopulations in spleen and lymph nodes.

c) Lipopolysaccharide and lipoprotein stimulate different B cell subpopulations of the spleen.

d) Lipopolysaccharide stimulated or lipoprotein‐stimulated B cells from lymph nodes differ from those in the spleen in that the latter show a markedly higher rate of labelling for GP 75, suggesting a higher rate of immunoglobulin M (IgM) synthesis.
Purification and Properties of Strictosidine Synthase, the Key Enzyme in Indole Alkaloid Formation
Authors: TREIMER Johannes F. , ZENK Meinhart H.
Published: March 2005

A new enzyme, strictosidine synthase, which catalyzes the synthesis of 3‐α(S)‐strictosidine from tryptamine and secologanin was isolated from the soluble protein extract of Catharanthus roseus cell suspension cultures and was purified approximately 50‐fold by ammonium sulfate fractionation, column chromatography on DEAE‐cellulose, Ultrogel AcA34 and isoelectric focusing. The apparent molecular weight of the enzyme was 34000. The pH optimum was 6.8, apparent Km values for tryptamine and secologanin were 2.3 mM and 3.4 mM respectively for the enzyme to synthesize strictosidine. Strictosidine synthase shows high substrate specificity. No apparent cofactor requirement could be demonstrated. Of several enzyme inhibitors tested, only p‐chloromercuribenzoate inhibited the enzyme. The enzyme was relatively stable and could be stored at −20°C for periods of up to 1 year without appreciable loss of catalytic activity. The enzyme was demonstrated to occur in suspension cultures of 15 different species belonging to 9 different genera of the indole‐alkaloidproducing subfamily Plumerioideae of the Apocynaceae family.

This enzyme is responsible for the synthesis of strictosidine the key intermediate in the formation of the majority of monoterpenoid indole alkaloids occurring in the plant kingdom.
Metal Activation of Galactosylhydroxylysyl Glucosyltransferase, an Intracellular Enzyme of Collagen Biosynthesis
Authors: MYLLYLÄ Raili , ANTTINEN Henrik , KIVIRIKKO Kari I.
Published: March 2005

Metal activation of galactosylhydroxylysyl glucosyltransferase was studied with EDTA‐treated highly purified enzyme from chick embryos and EDTA‐treated citrate‐soluble collagen substrate. Mn2+ activated the transferase at much lower concentrations than thought previously, and high Mn2+ concentrations were more effective than could be expected from the Michaelis‐Menten equation. The data suggest that the enzyme can bind at least two Mn2+ ions, the first having a Kd of 3–5 μM (site I) and the second 50–70 μM (site II). The binding of the second Mn2+ decreases the Km of UDP‐glucose from 25 μM to 6–7 μM, whereas the V at a saturating concentration of UDP‐glucose is unaffected. The EDTA‐treated enzyme possessed some activity even without added Mn2+, suggesting that either the transferase can be activated in the absence of any metal or that it has one additional metal binding site with high affinity. The kinetic data would be consistent with a reaction scheme involving an ordered binding of Mn2+, UDP‐glucose and the collagen substrate at low Mn2+ and an ordered binding of Mn2+, Mn2+, UDP‐glucose and the collagen substrate at high Mn2+ concentrations. In both cases the binding of the Mn2+, which occurs before that of UDP‐glucose, is at thermodynamic equilibrium. Zn2+, Cu2+, Cd2+, Ni2+ and Ca2+ are inhibitors of the Mn2+‐activated enzyme.

Fe2+ (Kd 5–7 μM) and Co2+ (Kd 30 μM) also activate the enzyme at low concentrations, whereas no further activation, but instead an inhibition, was found at high concentrations. Ca2+ and Mg2+, which have previously been reported to serve as metal co‐factors, did not activate the enzyme alone and did not stimulate the glucosyltransferase activated at low Mn2+ concentrations. The Kd values for Mn2+ at site II and for Co2+ are probably much higher than the physiological concentrations, whereas the activation by Mn2+ at site I and by Fe2+ may be of physiological significance.
Mössbauer Spectroscopic Studies of Iron in Proteus mirabilis
Authors: DICKSON Dominic P. E. , ROTTEM Shlomo
Published: March 2005

Mössbauer measurements of Proteus mirabilis cells grown in 57Fe‐enriched media show that considerable quantities of iron are incorporated, particularly in the stationary phase of growth. A major part of the iron is in a form which shows magnetically split spectra at low temperatures. Fractionation of cells reveals that iron is present in both the soluble and particulate fractions, with the larger proportion being found in the soluble fraction. The iron in the particulate fraction is predominantly found in the outer membrane.
Purification and Properties of Hydrogenase fromMegasphaera elsdenii
Authors: VAN DIJK Cees , MAYHEW Stephen G. , GRANDE Hans J. , VEEGER Cees
Published: March 2005

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacteriumMegasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain withMr∼ 50000 which contains 12 atoms of non‐haem iron and 12 atoms of acid‐labile sulphide. The enzyme is rapidly inactivated by O2and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (ε275 nm= 143 mM−1cm−1) and a shoulder between 350 nm and 400 nm (ε400 nm= 46 mM−1cm−1).

The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co‐operativity, but no indication of self‐association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin.

Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co‐operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one‐electron donors flavodoxin and methyl viologen semiquinone monomer and the two‐electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe‐4s) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer.

The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis‐Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two‐electron acceptors used are consistent with the proposed models, if it is assumed that the pathway followed is compulsory.

At this moment no choice can be made between the models proposed.
Chlorophyll‐Protein Complexes from Thylakoids of a Mutant Barley Lacking Chlorophyll b
Authors: WALDRON John C. , ANDERSON Jan M.
Published: March 2005

Modifications of the type and concentration of detergents and buffers used for thylakoid extraction and gel electrophoresis have provided improved resolution of chlorophyll‐protein complexes in mutant barley which lacks chlorophyll b. The use of sodium deoxycholate in the gels and in the solubilization of thylakoids reduced the amount of free chlorophyll significantly. Three chlorophyll‐a–protein complexes were resolved from mutant barley; two of these (CP1a and CP1) represent the reaction centre complex of photosystem 1, while the third complex, CPa, is the presumed reaction centre of photosystem 2. The fluorescence emission band of mutant barley complex CPa at 684 nm and the low fluorescence yield are consistent with expected characteristics of a reaction centre complex of photosystem 2. More chlorophyll (60 %) is associated with the reaction centre complex of photosystem 1 than with the presumed reaction centre complex of photosystem 2 (30%), and these amounts are double those found in the corresponding complexes of normal barley. It is proposed that the smaller photosynthetic unit of mutant barley thylakoids has twice as much chlorophyll a in photosystem 1 as in photosystem 2, and this chlorophyll is associated only with the two reaction centre complexes which are in direct contact. The sodium deoxycholate method may be useful for the resolution of pigment complexes from other thylakoids with inherently fragile photosystems.
Characterization of Preribosomal Ribonucleoprotein Particles from Tetrahymena pyriformis
Authors: RODRIGUES‐POUSADA Claudina , CYRNE Maria Luisa , HAYES Donal
Published: March 2005

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10–30% sucrose gradients.

After 1 min of labelling, the early ribosomal RNA precursor (36‐S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min).

The 80‐S particle contains 29‐S and 18‐S RNA species together with traces of 36‐S RNA; the 60‐S and 44‐S particles contain 26‐S and 17‐S RNAs respectively. Similar results were obtained when [Me‐3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30–70‐S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70–90‐S) components is still increasing after 10 min.

Only three types of ribonucleoprotein particles (80‐S, 66‐S, and 44‐S) were observed when the cells were labelled after prolonged starvation.

A scheme of ribosome biogenesis based on these results is presented.
Conformation of Cobrotoxin in Aqueous Solution as Studied by Nuclear Magnetic Resonance
Authors: ENDO Toshiya , INAGAKI Fuyuhiko , MIYAZAWA Tatsuo , HAYASHI Kyozo
Published: March 2005

The 270‐MHz proton NMR spectra of cobrotoxin from Naja naja atra were observed in 2H2O solution. The pKa value (5.93) of His‐32 is slightly lower than the pKa value (6.65) of the reference model of N‐acetylhistidine methylamide, because of the electrostatic interaction with Arg‐33 and Asp‐31. The pKa value (5.3—5.4) of His‐4 is appreciably low, because of the interaction with the positively charged guanidino group possibly of Arg‐59. The hydrogen‐deuterium exchange rates in 2H2O solution were measured of cobrotoxin and imidazole‐bearing models. The second‐order rate constants of N‐acetylhistidine methylamide, N‐acetylhistidine and imidazole acetic acid satisfy the Brønsted relation. With reference to this Brønsted relation, the imidazole ring of His‐32 is confirmed to be exposed. The imidazole ring of His‐4 is also exposed and the exchange rate is excessively promoted by the presence possibly of Arg‐59 in the proximity. All the methyl proton resonances are assigned to amino‐acid types, by conventional double‐resonance method and more effectively by the spin‐echo double‐resonance method. Eight methyl proton resonances are identified as due to the γ and/or δ‐methyl groups of Val‐46, Leu‐1, Ile‐50 and Ile‐52 residues. The proximity of aromatic ring protons and methyl protons is elucidated by the analyses of nuclear Overhauser effect enhance‐ ments. The aromatic proton resonances of Trp‐29 are affected by the ionizable groups of Asp‐31, His‐32 and Tyr‐35. The methyl groups of Ile‐50 are in the proximity to the aromatic ring of Trp‐29 and the methyl groups of Ile‐52 are in the proximity to Tyr‐25. The highest‐field methyl proton reso‐ nance is due to a threonine residue in the proximity to His‐4. The appreciable temperature‐dependent chemical shift of this methyl proton resonance suggests a temperature‐dependent local conformational equilibrium around the His‐4 residue of the first loop of the cobrotoxin molecule.
Cyanine Dye as Monitor of Membrane Potentials in Escherichia coli Cells and Membrane Vesicles
Authors: LETELLIER Lucienne , SHECHTER Emanuel
Published: March 2005

The fluorescence response of a positively charged cyanine dye: 3,3′‐dimethylindodicarbocyanine iodide can be specifically related to the generation in Escherichia coli cells and E. coli membrane vesicles of an electrical membrane potential induced either by substrate oxidation or by an artificially imposed potassium diffusion gradient. The energy‐dependent quenching of the dye fluorescence correlates well with the known effect on ΔΨ of: oxidation of various energy sources, external pH and solute accumulation. Thus, in the vesicles, the fluorescence quenching of the dye increases from succinate to d‐lactate, to ascorbate/phenazine methosulfate and parallels the increasing ability of these electron donors to generate a ΔΨ. In the vesicles, ΔΨ is only weakly dependent on external pH, whereas in the cells, ΔΨ increases with increasing external pH. Lactose accumulation in the vesicles results in the partial utilization of ΔΨ A calibration of the dye fluorescence in terms of ΔΨ has been determined using valinomycin‐induced potassium diffusion potential.
HeLa Cell Cytoplasmic mRNA Contains Three Classes of Sequences Predominantly Poly(A)‐Free, Predominantly Poly(A)‐Containing and Bimorphic
Authors: MILCAREK Christine
Published: March 2005

The mRNA species which exist in the HeLa cell polyribosomes in a form devoid of A sequences longer than 8 nucleotides constitute the poly(A)‐free class of mRNA. The rapidly labelled component of this mRNA class shares no measurable sequence homology with poly(A)‐containing RNA. If poly(A)‐free mRNA larger than 12 S labelled for 2 h in vivo is hybridized with total cellular DNA, it hybridizes primarily with single‐copy DNA. When a large excess of steady poly(A)‐containing RNA is added before hybridization of labelled poly(A)‐free RNA, no inhibition of hybridization occurs. This indicates the existence of a class of poly(A)‐free mRNA with no poly(A)‐containing counterpart.

Some mRNA species can exist solely as poly(A)‐containing mRNAs. These mRNAs in HeLa cells are found almost exclusively in the mRNA species present only a few times per cell (scarce sequences). Some mRNA species can exist in two forms, poly(A)containing and lacking, as evidenced by the translation data in vitro of Kaufmann et al. [Proc. Natl Acad. Sci. U.S.A. 74, 4801–4805 (1977)]. In addition, if cDNA to total poly(A)‐containing mRNA is fractionated into abundant and scarce classes, 47 % of the scarce class cDNA can be readily hybridized with poly(A)‐free mRNA. 10 of the abundant cDNA to poly(A)‐containing mRNA will hybridize with poly(A)‐free sequences very rapidly while the other 90% hybridize 160 times more slowly, indicating two very different frequency distributions. The cytoplasmic metabolism of these three distinct mRNA classes is discussed.
Evidence by Chemical Modification for the Involvement of One or More Tryptophanyl Residues of Bovine Antithrombin in the Binding of High‐Affinity Heparin
Authors: BJÖRK Ingemar , NORDLING Kerstin
Published: March 2005

Tryptophanyl residues of bovine antithrombin were modified with N‐bromosuccinimide at near‐neutral pH. The reaction was found to be specific for tryptophan at low levels of modification, i.e. when only up to 1–1.3 mol tryptophan/mol protein were oxidized. Further modification led to extensive side reactions. Modification of an average of about one tryptophanyl residue per protein molecule did not affect antithrombin activity measured in the absence of heparin, but decreased the activity assayed in the presence of heparin to about half the value given by unmodified antithrombin. Addition of an excess of high‐affinity heparin to a similarly modified antithrombin sample resulted in much smaller circular dichroism, ultraviolet absorption and fluorescence changes than those observed with the intact protein. Modification experiments in the presence of excess high‐affinity heparin gave a definitely lower extent of modification than when heparin was excluded. These studies thus reinforce the conclusion from previous spectroscopic analyses that one or more tryptophanyl residues of antithrombin are involved in the binding of high‐affinity heparin, presumably by being located at or close to the heparin binding site.
Structural Characterization by Nuclear Magnetic Resonance of a Reactive‐Site 13Carbon‐labelled Basic Pancreatic Trypsin Inhibitor with the Peptide Bond Arg‐39–Ala‐40 Cleaved and Arg‐39 Removed
Authors: RICHARZ Rene , TSCHESCHE Harald , WÜTHRICH Kurt
Published: March 2005

With the use of an enzymatic replacement method, 90%‐enriched [1‐13C]lysine was introduced into the reactive site of the basic pancreatic trypsin inhibitor. Characterization of the labelled inhibitor with 13C nuclear magnetic resonance (NMR), 1H NMR and chemical methods showed that while the reactive‐site peptide bond Lys‐15–Ala‐16 was properly resynthesized, the poly‐peptide chain was cleaved at the peptide bond Arg‐39–Ala‐40 and Arg‐39 was removed. Detailed 1H NMR studies showed further that, with the exception of the immediate environment of the modification site, the average spatial structure of the native inhibitor was preserved in the modified protein. Compared to the native inhibitor, the thermal stability of the globular conformation was found to be reduced, interior amide protons exchanged at a faster rate and the internal mobility of aromatic rings located outside the immediate environment of the cleaved peptide bond was essentially unchanged. These observations coincide closely with previous reports on different modifications of the inhibitor and can be explained by a recently proposed dynamic multi‐state model for globular proteins. Since the fundamental structural properties of the native inhibitor and full inhibitory activity are preserved after resynthesis, the[1‐13C]lys‐15‐labelled inhibitor with the peptide bond Arg‐39–Ala‐40 cleaved and Arg‐39 removed should be suitable for 13C NMR studies of mechanistic aspects of proteinase‐inhibitor interactions.
Structural Features of the Carbohydrate Units of Plasma Glycoproteins
Authors: FINNE Jukka , KRUSIUS Tom
Published: March 2005

For the characterization of the type and relative abundance of different carbohydrate structures in plasma glycoproteins, rat whole plasma was subjected to exhaustive proteolytic digestion and the resulting glycopeptides were fractionated into structurally district groups by chromatography on concanavalin‐A‐Sepharose followed by mild NaOH/NaBH4 treatment and gel filtration. The most abundant fraction was found to be the biantennary transferrin‐type glycopeptides (71% of recovered carbohydrate) followed by triantennary fetuin‐type structures and a smaller proportion of more branched structures (together 24%), neutral mannose‐rich chains (2%) and O‐glycosidic chains (3%). Structural similarity of the two major fractions to the transferrin and fetuin glycopeptides was supported by similar elution volumes in gel filtration, similar behaviour in concanavalin A affinity chromatography and similar sugar composition and substitution pattern of sugars in methylation analysis.

The glycopeptide pattern differs significantly from that observed previously for brain, kidney, liver or erythrocytes, where the more branched structures predominate, and which also contain higher amounts of the neutral mannose‐rich chains. As indicated by methylation analysis, the plasma glycoproteins are characterized by carbohydrate units terminating in N‐acetylneuraminic acid. The level of terminal galactose, N‐acetylglucosamine and fucose residues was low. This finding is in correlation with the described occurrence of binding‐activities in liver and other tissues which remove glycoproteins having these sugar terminals from the circulation.
Secondary Structure of the 5′ End of Bacteriophage MS2 RNA
Authors: ISERENTANT Dirk , FIERS Walter
Published: March 2005

To refine the secondary structure model of the 5′ end of the bacteriophage MS2 genome, 32P‐labeled MS2 RNA was partially digested with T1 RNase or with Cm‐RNase and the 5′‐end fragment was isolated, renatured and submitted to treatment with methoxyamine or kethoxal. The resulting modified RNA was digested with T1 RNase and the products were separated by minifingerprinting. Methoxyamine‐induced modification of exposed cytidines was detected by differential mobility of modified oligonucleotides, while kethoxal‐induced alteration of exposed guanosines was monitored by resistance to T1 ribonuclease digestion. The positions of the modified residues are discussed in terms of an improved secondary structure model proposed for the 5′ end of the viral RNA. The structure itself is discussed in relation to sequence conservation and biological function.
Aerobic Lactate Synthesis by Cardiac Muscle
Authors: STRONG Peter , MULLINGS Rosanne , ILLINGWORTH John A.
Published: March 2005

Cardiac lactate production under aerobic conditions is absolutely dependent upon the availability of extracellular pyruvate. In the steady state, aerobic lactate output is largely independent of cardiac work load, but increases slightly when octanoate is included in addition to pyruvate in the perfusion fluid. Transient episodes of supra‐normal lactate production are seen after sudden increases in cardiac work output, and also after transitions from octanoate to pyruvate in the perfusion media. These pulses of lactate production are invariably associated with the slow activation of pyruvate dehydrogenase in response to a sudden change in cardiac metabolic state, and they are abolished by pre‐perfusion with dichloroacetate, which converts pyruvate dehydrogenase into the fully active form. A second, additional component of the lactate pulses is sensitive to pre‐perfusion with the transaminase inhibitor aminooxyacetate. The size of the second component is markedly dependent upon the precise protocol adopted for the experiment, and these variations suggest that the second component is associated with a major redistribution of cardiac Krebs' cycle intermediates and amino acids following the initial exposure to pyruvate‐containing media. Steady‐state aerobic lactate production is insensitive to both dichloroacetate and aminooxyacetate, and is thought to result from a direct exchange of malate for oxaloacetate across the heart mitochondrial membranes.