bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on Polypeptide‐Chain‐Elongation Factors from an Extreme Thermophile, Thermus thermophilus HB8
Authors: ARAI Ken‐ichi , ARAI Naoko , NAKAMURA Shun , OSHIMA Tairo , KAZIRO Yoshito
Published: March 2005

Catalytic properties of the elongation factors from Thermus thermophilus HB8 have been studied and compared with those of the factors from Escherichia coli.

The formation of a ternary guanine‐nucleotide · EF‐Tu · EF‐Ts complex was demonstrated by gel filtration of the T. thermophilus EF‐Tu · EF‐Ts complex on a Sephadex G‐150 column equilibrated with guanine nucleotide. The occurrence of this type of complex has not yet been proved with the factors from E. coli.

The dissociation constants for the complexes of T. thermophilus EF‐Tu · EF‐Ts with GDP and GTP were 6.1 × 10−7 M and 1.9 × 10−6 M respectively. On the other hand, T. thermophilus EF‐Tu interacted with GDP and GTP with dissociation constants of 1.1 × 10−9 M and 5.8 × 10−8 M respectively. This suggests that the association of EF‐Ts with EF‐Tu lowered the affinity of EF‐Tu for GDP by a factor of about 600 and facilitated the nucleotide exchange reaction.

Although the T. thermophilus EF‐Tu · EF‐Ts complex hardly dissociates into EF‐Tu and EF‐Ts, a rapid exchange was observed between free EF‐Ts and the EF‐Tu · EF‐Ts complex using 3H‐labelled EF‐Ts. The exchange reaction was independent on the presence or absence of guanine nucleotides.

Based on the above findings, an improved reaction mechanism for the regeneration of EF‐Tu GTP from EF‐Tu · GDP is proposed.

Studies on the functional interchangeability of EF‐Tu and EF‐Ts between T. thermophilus and E. coli has revealed that the factors function much more efficiently in the homologous than in the heterologous combination.

T. thermophilus EF‐Ts could bind E. coli EF‐Tu to form an EF‐Tu (E. coli) · EF‐Ts (T. thermophilus) hybrid complex. The complex was found to exist in a dimeric form indicating that the property to form a dimer is attributable to T. thermophilus EF‐Ts. On the other hand, no stable complex between E. coli EF‐Ts and T. thermophilus EF‐Tu has been isolated.

The uncoupled GTPase activity of T. thermophilus EF‐G was much lower than that of E. coli EF‐G. T. thermophilus EF‐G formed a relatively stable binary EF‐G · GDP complex, which could be isolated on a nitrocellulose membrane filter. The Kd values for EF‐G · GDP and EF‐G · GTP were 6.7 × 10−7 M and 1.2 × 10−5 M respectively. The ternary T. thermophilus EF‐G · GDP · ribosome complex was again very stable and could be isolated in the absence of fusidic acid. The stability of the latter complex is probably the cause of the low uncoupled GTPase activity of T. thermophilus EF‐G.
Control of Proton Conduction by the H+‐ATPase in the Inner Mitochondrial Membrane
Authors: PANSINI Angela , GUERRIERI Ferruccio , PAPA Sergio
Published: March 2005

Proton conduction in submitochondrial particles with various degrees of resolution of the H+‐ATPase has been studied.

Proton conduction was analyzed by following the kinetics of the anaerobic decay of protons taken up by submitochondrial particles during respiratory pulses.

In EDTA submitochondrial particles proton release exhibited biphasic kinetics; both phases were depressed by collapsing aerobic Δφ.

The slow phase of proton diffusion was inhibited by oligomycin and N,N′‐dicyclohexylcarbodiimide as well as by F1 ligands like adenylyl 5′‐imidophosphate and alkyl cations. Inhibition of proton conduction by F1 ligands exhibited a sigmoidal titration curve and was synergistic with inhibition by oligomycin. Removal of the ATPase inhibitor markedly enhanced the kinetic constant of the slow proton diffusion process.

The initial rapid phase of proton diffusion was inhibited by N,N′‐dicyclohexylcarbodiimide and oligomycin but was unaffected by F1 ligands.

When the F1 moiety was removed from the particles, proton conduction became monophasic and was not any more inhibited by F1 ligands.

It is concluded that the H+‐ATPase contributes to both phases of proton conduction and that energy‐linked cooperative interaction of the F1 with the F0 moiety controls proton conductivity.

Evidence is also providëd for involvement of negatively charged groups in hydrophobic environments of the F1 moiety in proton translocation by the H+‐ATPase.
Electrophoretic Fractionation and Translation in vitro of Poly(rA)‐Containing RNA from Maize Endosperm
Authors: WIENAND Udo , FEIX Günter
Published: March 2005

In the search for individual mRNAs coding for particular zein proteins, polysomal RNA was isolated from the endosperm of 22‐days post‐pollination maize kernels. This RNA was enriched for poly(rA)‐containing RNAs and then submitted to preparative polyacrylamide gel electrophoresis under denaturing conditions. After electrophoresis, RNA fractions were eluted from the polyacrylamide gel and analyzed for zein mRNA activity by translation in vitro in the wheat germ system. The wheat germ system had previously been optimized for accurate translation of zein mRNAs. By a gel‐electrophoretic analysis of immunoprecipitated products from the trnaslation reactions in vitro, it could conclusively be shown that the endosperm of developing maize kernels contains two separable mRNAs for zein, one coding for the 22000‐Mr protein and one coding for the 19000‐Mr protein.
Fluorescence Properties of Reduced Flavins and Flavoproteins
Authors: VISSER Antonie J. W. G. , GHISLA Sandro , MASSEY Vincent , MÜLLER Franz , VEEGER Cees
Published: March 2005

Fluorescence lifetimes and polarized emission properties of reduced flavin were measured using several model compounds and flavoproteins. Depending on the conditions of solvent and temperature or reduction method the lifetimes vary between 1 and 15 ns. The longer lifetime values are found in several forms of reduced lactate oxidase, in which a good correlation exists between fluorescence intensity and lifetime. In practically all flavoproteins the fluorescence is heterogeneous. Several mechanisms are proposed to explain the observed heterogeneity in lifetimes. The reduced models in glycerol at subzero temperature exhibit high degrees of polarization of the fluorescence, whereas distinct depolarization is encountered in several reduced flavoproteins suggesting a certain mobility of the flavin chromophor.
Release of a Two‐Chain Form of Antithrombin from the Antithrombin‐Thrombin Complex
Authors: FISH Wayne W. , BJÖRK Ingemar
Published: March 2005

The stable, inactive complex between antithrombin and thrombin was prepared and subsequently purified free of reactants by a procedure which minimized secondary thrombin proteolysis. Dissociation of the complex was effected by hydroxylamine or ammonia in the presence or absence of sodium dodecyl sulphate, or by prolonged treatment at 100°C in the presence of dodecyl sulphate. All these procedures released from the complex a proteolytically modified, two‐chain form of antithrombin and not the intact protein. Dissociation in the absence of denaturant also released active thrombin. Control experiments indicated that the inhibitor was indeed released from the complex as the modified form of antithrombin rather than as intact antithrombin which was subsequently modified by the dissociation agents or by thrombin. The modified form of antithrombin dissociated from the complex could not be differentiated electrophoretically from a proteolytically modified, inactive form of antithrombin which has been shown previously to be produced free in solution during the reaction between antithrombin and thrombin. These observations suggest that limited proteolysis of antithrombin by thrombin may be involved in the formation of the inactive complex. The inhibitor may thus exist in the complex as a proteolytically modified, two‐chain species. However, a state in which the scissilc peptide bond of the inhibitor only forms a tetrahedral adduct with the active site serine of the enzyme, as shown by X‐ray crystallography in other protease‐protease inhibitor complexes, cannot be excluded.
Nucleotide Sequences Adjacent to the Proteins Covalently Linked to the Cowpea Mosaic Virus Genome
Authors: STANLEY John , VAN KAMMEN Albert
Published: March 2005

The sequences of the first 17 nucleotides of cowpea mosaic virus middle and bottom RNAs adjacent to the covalently‐linked proteins have been determined. Sequences of the oligonucleotides, produced by complete T1 RNase digestion, were established after labelling of the 3′ termini in vitro using RNA ligase. Both sequences are A/U‐rich, the first nine nucleotides being identical.
Liver Phosphorylase b Kinase
Authors: VANDENHEEDE Jackie R. , DE WULF Henri , MERLEVEDE Wilfried
Published: March 2005

Phosphorylase b kinase was extensively purified from rat liver. It was isolated in a form which could be activated 20–30‐fold by a preincubation with adenosine 3′ : 5′‐monophosphate (cyclic AMP) and ATP‐Mg. This activation was time‐dependent, and was paralleled by a simultaneous incorporation of 32P from [γ‐32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the α and β subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio‐Gel A‐50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 × 106. After activation, the most purified liver preparation had a specific activity about 10‐fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly activeabove neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis‐(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.
Mavicyanin, a Blue Copper Protein from Cucurbita pepo medullosa
Authors: MARCHESINI Augusto , MINELLI Martin , MERKLE Hellmut , KRONECK Peter M. H.
Published: March 2005

The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa.

Mavicyanin contains one type‐1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (ɛ= 5000 M−1 cm−1/Cu, A280/A600= 8.0 ± 0.5, A600/A403= 7.0 ± 0.25, maximum of fluorescence emission at 335 nm.

In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz= 2.287, gy= 2.077, gx= 2.025, Az= 3.5 mT, Ay= 2.9 mT and Ax= 5.7 mT.

Like other simple type‐1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicy‐anin is readily reduced by hydroquinone or L‐ascorbic acid. Its midpoint potential E′m was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.
Methylation Sites in HeLa Cell Ribosomal Proteins
Authors: SCOLNIK Pablo A. , ELICEIRI George L.
Published: March 2005

The methylation of HeLa cell ribosomal proteins has been studied by paper electrophoresis separation of the peptides produced after trypsin digestion of individual ribosomal proteins. Methylation in vivo was detected by 3H labeling of peptides from cells that had been incubated with a mixture of [methyl‐3H]methionine plus [35S]methionine, the incorporation of the 3H‐labeled amino acid methionine being corrected by the 35S uptake. Each methylated peptide thus represented at least one methylation site (one or more methylated amino acids and their adjacent amino acid sequence). Eleven ribosomal proteins were found to be methylated. As many as five methylation sites were present in one ribosomal protein. In several ribosomal proteins some sites were methylated when ribosome synthesis was suppressed with low levels of actinomycin D, while other sites were not. Therefore, some of the sites in a given protein were apparently methylated in mature ribosomes, while other sites were methylated during ribosome processing. The methyl‐group‐specific labeling of some sites, but not others, was suppressed by cycloheximide, thus suggesting that the methylation of some sites was dependent on protein synthesis.

A cell‐free system from cultured HeLa cells is described, which reproduces the methylation pattern of ribosomal proteins in vivo in two ways: methylation was observed both in vivo and in vitro (a) in the same ribosomal proteins; and (b) in the same methylation sites of a given ribosomal protein. In addition, our data in vitro suggest that the methylation of some human ribosomal protein sites may involve a methyl donor other than S‐adenosylmethionine.
Effect of Tunicamycin on Transport of Lysosomal Enzymes in Cultured Skin Fibroblasts
Authors: VON FIGURA Kurt , REY Martin , PRINZ Roswitha , ULLRICH Kurt , VOSS Bruno
Published: March 2005

After an initial lag phase, tunicamycin (0.12–3.6 μM) inhibits the secretion of β‐N‐acetylglucosaminidase by human skin fibroblasts. The inhibition persists for up to 30 h. Thereafter a more than fivefold‐increased rate of secretion is observed for at least 4 days. The intracellular β‐N‐acetylglucos aminidase activity decreases within 9 days in the presence of 0. 36 μM tunicamycin to 55% of that of controls. β‐N‐Acetylglucosaminidase formed in the presence of tunicamycin is only poorly recognized by fibroblasts, suggesting that tunicamycin interferes with the formation of the phosphomannosyl residues that function on β‐N‐acetylglucosaminidase as a recognition marker for the cell surface receptors present on fibroblasts. Tunicamycin (0.36–3.6 μM) inhibits endocytosis of exogenous lysosomal enzymes only after a preincubation of 24 h in the presence of the drug. Control experiments indicated that this inhibition is not due to a general inhibition of receptor‐mediated endocytosis. Incubation with tunicamycin leads furthermore to a loss of cell‐surface‐associated lysosomal enzymes.

These findings indicate that tunicamycin interferes with the expression of phosphomannose receptors on the cell surface of fibroblasts and the synthesis of the phosphomannosyl residues on lysosomal enzymes after an initial lag phase. These alterations are associated with an increased accumulation of β‐N‐acetylglucosaminidase extracellularly, a loss of that enzyme from intracellular sites and from the cell surface. A similar mislocation of lysosomal enzymes in mucolipidosis II fibroblasts that is associated with a deficiency of the recognition marker on lysosomal enzymes but normal expression of the phosphomannose receptors led to the hypothesis that lysosomal enzymes undergo a secretion‐endocytosis process during their transfer into lysosomes [Hickman, S. & Neufeld, E. F. (1972) Biochem. Biophys. Res. Commun. 49, 992–999]. The present results suggest that phosphomannosyl residues within N‐glycosidically linked oligosaccharide chains on lysosomal enzymes [von Figura, K. & Klein, U. (1979) Eur. J. Biochem. 94. 347–354] function either intracellularly or on the cell surface as signal for their transfer into lysosomes.
The Involvement of Poly(ADP‐ribose) Polymerase in the Degradation of NAD Caused by γ‐Radiation and N‐Methyl‐N‐Nitrosourea
Authors: SKIDMORE Christopher J. , DAVIES Margaret I. , GOODWIN Patricia M. , HALLDORSSON Haraldur , LEWIS Pamela J. , SHALL Sydney , ZIA'EE Abed‐Ali
Published: March 2005

Both N‐methylN‐nitrosourea and γ‐radiation lower cellular NAD in mouse leukaemia cells (L‐1210) in a dose‐dependent way. The minimum NAD level is reached 2 h after a brief exposure to N‐methyl‐N‐nitrosourea, but within 15 min of y‐irradiation. The cells remain metabolically active; they are able to recover their control NAD levels and are impermeable to trypan blue.

Several inhibitors of poly(ADP‐ribose) polymerase inhibit the drop in cellular NAD caused by these two agents: 2 mM 5‐methylnicotinamide, 1 mM theophylline or 1 mM theobromine inhibit the effect of N‐methyl‐N‐nitrosourea on cellular NAD level; 200 uM thymidine, 500 uM 5‐methyl‐nicotinamide, 500 αM theophylline and 500 αM theobromine prevent the lowering of cellular NAD by y‐irradiation. The extent to which the drop in cellular NAD is inhibited is dependent on both the concentration of cytotoxic agent and of polymerase inhibitor. Caffeine will inhibit the drop in NAD but only at 10 mM, while nicotonic acid is ineffective even at this dose.

The activity of poly(ADP‐ribose) polymerase in permeabilized cells immediately after y‐radiation increases with dose up to 12 krad, giving a maximal 3.4‐fold stimulation of the enzyme activity, whereas the degradation of NAD under conditions optimal for NAD glycohydrolase does not change. The activity of the polymerase shows a close temporal correlation with the NAD drop following both γ‐radiation and N‐methyl‐N‐nitrosourea. The enzyme activity is maximal when the NAD content is decreasing at the highest rate and has returned to normal levels when it ceases falling.

In permeabilized cells we can distinguish poly(ADP‐ribose) polymerase and NAD glycohydrolase activity by their differential response to inhibitors. The polymerase is sensitive to 5‐methylnicotin‐amide, theophylline, theobromine and thymidine; the NAD glycohydrolase is sensitive to 5‐methyl‐nicotinamide and theophylline, but not to theobromine and thymidine.

We propose that the decrease in cellular NAD level produced by y‐radiation and by N‐methyl‐N‐nitrosourea is caused by an increased flux through poly(ADP‐ribose) mediated by an increased activity of poly(ADP‐ribose) polymerase. This consequently lowers the cellular NAD level. This hypothesis implies an involvement of (ADP‐ribose)n in the cellular response to cytotoxic drugs.
Isolation of Synaptosomal Plasma Membranes from Cholinergic Nerve Terminals and a Comparison of Their Proteins with Those of Synaptic Vesicles
Authors: STADLER Herbert , TASHIRO Tomoko
Published: March 2005

Plasma membranes were purified from purely cholinergic nerve endings (synaptosomes) isolated from the electric organ of Torpedo marmorata. Synaptosomes were lysed, membranes recovered and further separated by density gradient centrifugation. A fraction was obtained enriched in 5′‐nucleotidase, Na+, K+‐activated ATPase and acetylcholine esterase. Morphological examination showed abundant membrane fragments of the size range of synaptosomes and few of vesicle size. The fraction has a characteristic protein composition upon gel electrophoresis. Five reproducible major bands with apparent Mr of 100000, 75000, 52000, 42000 and 35000–33000 are found. A gel‐electrophoretic comparison with proteins from synaptic vesicles from the same source (major bands Mr, 160000, 147000, 42000, 34000 and 25000) was made. Comigration of major bands was detected in one‐dimensional gel electrophoresis the 42000‐Mr components. Upon two‐dimensional gel electrophoresis the 42000‐Mr component comigrates with a similar component in vesicles, recently characterized as actin; the other components are different. The presence of tubulin‐like polypeptides is unlikely.

Beside actin, all major vesicle proteins are often detected in small amounts in the plasma membrane preparation. It cannot be decided if they result from fused or contaminating vesicle membranes, but since they are essentially absent in some preparations, it seems that the plasma membrane does not contain vesicle proteins.
Hepatic Glutamine Metabolism under the Influence of the Portal Ammonia Concentration in the Perfused Rat Liver
Authors: HÄUSSINER Dieter , SIES Helmut
Published: March 2005

l‐Glutamine, when added to the portal perfusate in a system of isolated perfused rat liver (non‐recirculating open system) at physiological concentration of 0.6 mM, does not lead to an extra production of urea and ammonia, but it does so when ammonium ions at physiological concentration of 0.3 mM are simultaneously present. This stimulatory effect of ammonium ions on glutaminase activity is readily reversible. The effect of portal ammonium ions is half‐maximal at 0.2–0.3 mM and maximal at about 0.6 mM.

The maximal rate of production of urea‐N plus ammonia from glutamine is exhibited at 10–12 mM glutamine, amounting to about 2.6 μmolxmin−1 xg wet weight−1.

The modulatory effects of ammonium ions on the disposition of glutamine by the liver were demonstrated (a) by the extra release of nitrogen from glutamine, (b) by the release of 14CO2 from [U‐14C]glutamine, and (c) by measurement of glutamine uptake.

The simultaneous operation of glutamine synthetase and glutaminase activities in liver was demonstrated to exist (futile cycle), based on 14CO2 release from [U‐14C]glutamine and the positive glutamine balance across the hepatic and portal veins, and further on the effects of methionine sulfoximine. This inhibitor of glutamine synthetase abolished net glutamine release and left the rate of 14CO2 relcase from [U‐14C]glutamine unaltered. Moreover, there is an extra O2 uptake by the liver upon glutamine addition which is not accounted for by extra urea synthesis; the extra O2 uptake increases with the portal glutamine supply. An estimate of the rate of this futile cycle at 0.6 mM glutamine is given, approx. 0.1 μmol × min−1× g wet wt liver−1.

The stimulatory effect of portal ammonium ions on hepatic glutamine breakdown is discussed in terms of an inter‐organ feed‐forward mechanism, e. g. between small intestine and liver during conditions of increased glutamine supply and, therefore, increased requirement of glutamine removal from the circulation.
Nuclear Poly(A) Polymerase Activities in the Rabbit Uterus
Authors: ORAVA Mauri M. , ISOMAA Veli V. , JÄNNE Olli A.
Published: March 2005

Poly(A) polymerase activities were measured from intact rabbit uterine nuclei and chromatin after a single dose of progesterone, and following a 5‐day treatment with progesterone alone or in combination with estradiol. A single intravenous dose of progesterone (5 mg/kg) to estrogen‐primed rabbits elicited an early increase in both bound and free nuclear poly(A) polymerase activities, the peak activity (about 40% over the controls) being reached 30–60 min posttreatment. The bound enzyme activity seemed to exhibit a second rise that occurred 12 h after progesterone administration. A 5‐day treatment of estrous rabbits with progesterone (1 mg/kg) brought about a sixfold elevation in the bound poly(A) polymerase and a twofold increase in the free nucleoplasmic enzyme activity. A corresponding activation also occurred in the chromatin‐associated poly(A) polymerase. Concomitant administration of estradiol (50 μg/kg) with progesterone significantly depressed progesterone‐elicited increases in the poly(A) polymerases. Changes in the nuclear RNA polymerase I and II were minor during a long‐term progesterone treatment, while the total chromatin template activity, as measured in a transcription system in vitro using wheat germ RNA polymerase II, was significantly reduced after a 5‐day progesterone administration. When estradiol was given along with progesterone, it counteracted the effect of progesterone on the chromatin template.

These results suggest that progesterone exerts in the rabbit uterus posttranscriptional actions on the metabolism and processing of mRNA species. Moreover, the antiprogestational action of estrogens was already noted in the regulation of enzymes responsible for completion of mature mRNA synthesis.
Degradation of Newly Synthesized Ribosomal Proteins and Histones in Regenerating Rat Liver with and without Treatment with a Low Dose of Actinomycin D
Authors: TSURUGI Kunio , OGATA Kikuo
Published: March 2005

The metabolism of newly synthesized ribosomal proteins in regenerating livers of rats pretreated with a low dose of actinomycin D which selectively inhibits rRNA synthesis, has been investigated. After 15 min of labeling most newly synthesized proteins were found in the cell sap of regenerating livers of rats pretreated for 1 h with actinomycin D and disappeared from this fraction during a subsequent chase in the presence of cycloheximide. However, no proteinase activity for ribosomal proteins was detected in cell sap. After treatment in vivo with E‐64, a thiol‐proteinase inhibitor which inhibits the ribosomal protein‐hydrolysing activities of nuclear and mitochondrial plus lysosomal fractions of rat liver, a fraction of newly synthesized ribosomal proteins was found to accumulate in nuclci of actinomycin‐d‐prctreated regenerating rat liver. These results may indicate that ribosomal proteins synthesized in the absence of rRNA synthesis accumulated in cell sap, and were then transported into nuclei where they were degraded by a proteinase sensitive to E‐64. In the absence of actinomycin D pretreatment smaller amounts of ribosomal proteins accumulated in nuclei of regenerating livers of E‐64‐treated rats. Furthermore, histones (except histone H1) also accumulated in nuclei of regenerating livers of E‐64‐treated rats with and without actinomycin D pretreatment. Thus, these two kinds of proteins, especially histones, are synthesized in excess of nascent rRNA or DNA, and are degraded preferentially in nuclei in regenerating rat liver.
Increased Urinary Excretion of Free N‐Acetylneuraminic Acid in Thirteen Patients with Salla Disease
Authors: RENLUND Martin , CHESTER M. Alan , LUNDBLAD Arne , AULA Pertti , RAIVIO Kari O. , AUTIO Seppo , KOSKELA Sirkka‐Liisa
Published: March 2005

Thirteen severely retarded patients with Salla disease, a new type of lysosomal storage disorder, have been studied biochemically. All patients excreted approximately ten times more free sialic acid than normal individuals. The isolated sialic acid was characterized by paper chromatography, thin‐layer chromatography, optical rotation, 13C and 1H nuclear magnetic resonance spectroscopy, and mass spectrometry of its permethylated derivative. The results clearly indicated that the excreted sialic acid was identical to N‐acetylneuraminic acid. The main sialylated trisaccharide present in the urine of the patients was identified as 3′‐sialyllactose by sugar and methylation analysis. The excreted amounts were found to be within normal range.
Comparison of Poly(A)‐Containing RNAs in Different Cell Types of the Lower Eukaryote Schizophyllum commune
Authors: ZANTINGE Bert , DONS Hans , WESSELS Joseph G. H.
Published: March 2005

Poly(A)‐containing RNAs were isolated from morphologically different cells of the fungus Schizophyllum commune. Using mRNA markers the number‐average length of poly(A)‐containing RNA in total RNA and in purified poly(A)‐containing RNA was estimated as 1100 nucleotides. Number‐average length of poly(A)‐tracts was 33 nucleotides. 2.5% of total RNA is poly(A)‐containing RNA and probably up to 7.5% are non‐polyadenylated polydisperse RNA sequences.

Saturation hybridization of poly(A)‐containing RNA to gap‐translated [3H]DNA resulted in 16% of the reactive single‐copy DNA to become S1 nuclease resistant. It was found that purified poly(A)‐containing RNA represented the entire RNA complexity, i. e. 10000 different RNA sequences in S. commune.

RNA sequences isolated from morphologically different mycelia and from fruiting and non‐fruiting mycelia were identical for at least 90%.
Improvements in Immunoprecipitation of Specific Messenger RNA
Authors: PAYVAR Farhang , SCHIMKE Robert T.
Published: March 2005

We have described previously procedures for the isolation of specific mRNA employing immunoprecipitation of polysomes. In spite of our success with ovalbumin mRNA in the chicken oviduct, we have had considerable difficulties in applying these same published techniques to the immunopurification of conalbumin mRNA, despite the fact that the chicken oviduct synthesizes up to 10% of protein as conalbumin. Here we describe a number of modifications and refinements which have proved essential in obtaining intact conalbumin mRNA in high purity and high yields. These refinements include: (a) improved purification of conalbumin in order to remove contaminating proteins that result in impure antibodies; (b) improved isolation of specific conalbumin antibody in high yields; (c) improved methods for reducing contamination by non‐specific polysomes; (d) improved techniques for isolation of RNA from immunoprecipitates resulting in less degradation and higher recovery of conalbumin mRNA; (e) improved techniques for efficient translation of conalbumin mRNA involving treatment of the RNA with methylmercury prior to translation.

We conclude that problems involved in the immunoprecipitation of different mRNAs may differ, and that various refinements in techniques may be required for obtaining highly purified preparations of intact mRNA in high yields.
Purification and Primary Structure of Cytochrome c‐552 from the Cyanobacterium, Synechococcus PCC 6312
Authors: AITKEN Alastair
Published: March 2005

Cytochrome c‐552 (soluble ‘cytochrome f) from the unicellular cyanobacterium Synechococcus PCC 6312 (ATCC 27167) was purified and the primary structure determined. The proposed sequence conists of one polypeptide chain of 87 residues. The sequence was determined by a combination of chemical and enzymatic cleavage, manual and automatic sequencing and mass spectroscopy. This is the first amino acid sequence of this cytochrome from a unicellular cyanobacterium to be determined in a study of the variation in primary structure between phylogenetically distant cyanobacteria. The sequence is compared to the primary structures of the cytochrome from filamentous cyanobacteria and from eukaryotic algae. The significance of these sequence comparisons to the current hypotheses concerning the origin of eukaryotic cells and their chloroplasts is discussed.
Mechanism of O‐Specific Polysaccharide Biosynthesis in Salmonella Serogroups C2 and C3
Authors: SHIBAEV Vladimir N. , DRUZHININA Tatiana N. , POPOVA Alla N. , ROZHNOVA Sophia Sh. , KILESSO Valentina A.
Published: March 2005

Cell envelope and soluble glycosyl transferase preparations from Salmonella newport (sero‐group C2) and Salmonella kentucky (serogroup C3) were found to catalyze formation of polyprenyl pyrophosphate tetrasaccharides corresponding to the structure of the repeating unit of the main chain of O‐specific polysaccharides. Plant polyprenyl phosphate may serve as an exogenous sugar acceptor. Galactose residue is an initiator of a chain growth: transfer of galactosyl phosphate from uridine diphosphate galactose onto the acceptor is followed by two consecutive mannosyl transfers from guanosine diphosphate mannose and rhamnosyl transfer from thymidine diphosphate rhamnose. Uridine diphosphate glucose and polyprenyl phosphate are converted by the enzyme preparations into polyprenyl monophosphate glucose which may transfer a glucosyl residue onto the polyprenyl pyrophosphate oligosaccharides. The resulting pentasaccharide derivatives may be polymerised by enzymes present in cell envelope preparations. The significance of these results for the understanding of the mechanism of O‐specific polysaccharide biosynthesis is discussed.
A Toxin Purified from the Venom of Black Widow Spider Affects the Uptake and Release of Radioactive γ‐Amino Butyrate and N‐Epinephrine from Rat Brain Synaptosomes
Authors: GRASSO Alfonso , SENNI Maria Immacolata
Published: March 2005

A purified neurotoxin from the venom of black widow spider Latrodectus mactans tredecimguttatus, when tested on synaptosomes by means of a perfusion technique, stimulates the release of previously accumulated radioactive transmitters.

Black widow spider toxin has also been shown to be a potent inhibitor of the uptake systems for γ‐aminobutyrate and N‐epinephrine. The release from synaptosomes of radioactive N‐epinephrine and γ‐aminobutyrate was rapidly increased by the presence of a low concentration of toxin (∼ 1 nM). The toxin does not appear to cause either non‐specific membrane damage or the release of a non‐transmitter substance like γ‐amino[14C]isobutyric acid.

The venom‐induced release of γ‐aminobutyrate was shown not to be dependent on extracellular calcium concentration provided that sodium ions are present. By contrast, that of N‐epinephrine appeared to be Ca‐dependent. The release of γ‐aminobutyrate induced by the toxin is not modified by the presence in the perfusing fluid of tetrodotoxin, dithioerythritol and strontium ions, or by pretreatment of synaptosomes with diaminobutyrate. The mechanism of toxin‐induced release is different from that of a depolarization‐induced release.

By correlating previous electrophysiological observations with the results obtained in this biochemical study it may be suggested that the toxin can be used in studies aimed at the identification of components specific to the presynaptic terminal membrane involved in neurotransmitter release.
1,4‐α‐Glucan Phosphorylase from the Slime Mold Physarum polycephalum
Authors: NADER Werner , BECKER Jörn‐Ullrich
Published: March 2005

Glycogen phosphorylase from macroplasmodia of Physarum polycephalum was purified 76‐fold to homogeneity. The native enzyme migrated as a single protein band on analytical disc gel electrophoresis coinciding with phosphorylase activity. After reduction in the presence of sodium dodecylsulfate one protein band was detectable which corresponded to an Mr, of 93000. The molecular weight of the native enzyme determined by gel sieving or gradient‐polyacrylamide gel electrophoresis was 172000 and 186000, respectively. The enzyme contained about 1 mol pyridoxal 5′‐phosphate and less than 0.1 mol covalently bound phosphate per mol subunit. The amino acid composition of the enzyme was determined. In the direction of phosphorolysis the kinetic data were determined by initial velocity studies, assuming a rapid equilibrium random mechanism. Glucose 1‐phosphate and GDP‐glucose were competitive inhibitors toward phosphate and non‐competitive to glycogen. 5′AMP, a weak activator of the enzyme, counteracted the glucose‐1‐phosphate inhibition completely. Physarum phosphorylase was compared with phosphorylases from other sources on the basis of chemical and kinetic properties. No evidence for the presence of phosphorylated forms has yet been found.
Purification and Properties of a Phosphorylase (Phosphoprotein) Phosphatase Associated with an Alkaline Phosphatase of Mr 35000 from Bovine Adrenal Cortex
Authors: LI Heng‐Chun
Published: March 2005

A metal‐ion‐independent, nonspecific phosphoprotein phosphatase (Mr= 35000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p‐nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ > Mn2+ > Co2+) and a sulfhydryl compound for activity, has been found to co‐purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal‐ion‐dependent form which, subsequently, can be reactivated by Co2+ > Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.
Inhibition of DNA Synthesis in Mammalian Cells by Daunomycin
Authors: SCHELLINX Jean A. A. , DIJKWEL Peter A. , WANKA Friedrich
Published: March 2005

The effect of the intercalating agent daunomycin on DNA synthesis was studied in cultured bovine liver cells. At low daunomycin concentrations (1 and 2 μM) the rate of [3H]thymidine incorporation decreased progressively with the duration of exposure to the inhibitor. This was accompanied by a shift of nascent DNA intermediates of replicon size to higher sedimentation values on sucrose gradients, indicating that daunomycin preferentially affects the initiation of replicating units, both in asynchronous and synchronized cells.

At high daunomycin concentrations (12 μM) the rate of chain growth was also markedly reduced. This was indicated by a rapid and nearly complete cessation of the [3H]thymidine incorporation and an accumulation of nascent DNA intermediates of low molecular weight. These observations are discussed in relation to a pre‐fork mode of DNA synthesis.
Androgenic Regulation of Messenger RNA Sequence Complexity in Accessory Sexual Tissues of the Male Rat Studied with Fractionated Complementary DNA
Authors: HIGGINS Stephen J. , FULLER Frances M. , JACKSON Philip J. , PARKER Malcolm G.
Published: March 2005

Effects of androgens on mRNA sequence complexity in the rat seminal vesicle have been investigated using complementary DNA fractionated on the basis of sequence abundance. Total cDNA complementary to poly(A)‐rich RNA from normal rats was hybridised with an excess of the same RNA to controlled r0t values and then the free cDNA was separated from cDNA · RNA hybrids by hydroxyapatite chromatography. Three cDNA fractions were obtained with very different hybridisation characteristics. Abundant cDNA hybridised to an excess of its parental RNA with an r0t1/2 of 2.46 × 10−3 mol 1−1 s and is complementary to about six or seven average‐sized sequences. Use of hybrid‐arrested translation in a cell‐free protein‐synthesising system has shown that this class of mRNA includes mRNAs coding for major androgen‐dependent secretory proteins. Moderate and scarce cDNA fractions each showed more complex hybridization kinetics; computer analysis suggested each is complementary to two groups of average‐sized sequences. Each cDNA fraction was hybridised to excess poly(A)‐rich RNA from normal or castrated rats and the kinetics compared. Castration had no effect on the total number of sequences present in any class and did not alter the relative concentration of the scarce sequences. A small (threefold) decrease was seen in the concentration of abundant sequences with a larger (tenfold) decrease in the moderate class. Both decreases were reversed by testosterone in vivo. The results are consistent with earlier studies where the effects of testosterone on seminal vesicle mRNA were followed using a translation assay and confirm that no gross differential effects are exerted on abundant mRNA coding for major secretory proteins. The cDNA fractions were also used to investigate the overlap in genetic expression between seminal vesicle and ventral prostate. Both tissues share all the scarce sequences in the same relative abundance. Less than 0.0015% and 0.004%, of prostatic mRNA is complementary to seminal vesicle abundant and moderate sequences respectively. Similarly prostatic abundant sequences account for less than 0.004% of seminal vesicle mRNA.
Ligand Competition Curves as a Diagnostic Tool for Delineating the Nature of Site‐Site Interactions: Theory
Authors: HENIS Yoav I. , LEVITZKI Alexander
Published: March 2005

A few molecular models have been developed in recent years to explain the mechanism of cooperative ligand binding. The concerted model of Monod, Wyman and Changeux and the sequential model of Koshland, Némethy and Filmer were formulated to account for positively cooperative binding. The pre‐existent asymmetry model and the sequential model can account for negatively cooperative ligand binding. In most cases, however, it is virtually impossible to deduce the molecular mechanism of ligand binding solely from the shape of the binding isotherm. In the present study we suggest a new strategy for delineating the molecular mechanism responsible for cooperative ligand binding from binding isotherms. In this approach one examines the effect of one ligand on the cooperativity observed in the binding of another ligand, where the two ligands compete for the same set of binding sites. It is demonstrated that the cooperativity of ligand binding can be modulated when a competitive ligdnd is present in the protein‐ligand binding mixture. A general mathematical formulation of this modulation is presented in thermodynamic terms, using model‐ independent parameters. The relation between the Hill coefficient at 50% ligand saturation with respect to ligand X in the absence, h(x), and in the presence of a competing ligand Z, h(x,z), is expressed in terms of the thermodynamic parameters characterizing the binding of the two ligands. Then the relationship between h(x) and h(x,z), in terms of the molecular parameters of the different allosteric models, is explored. This analysis reveals that the different allosteric models predict different relationships between h(x,z) and h(x). These differences are especially focused when Z binds non‐cooperatively. Thus, it becomes possible, on the basis of ligand binding experiments alone, to decide which of the allosteric models best fits a set of experimental data.
On the Structure of the Teichoic Acid from the Cell Wall of Streptomyces antibioticus 39
Authors: SHASHKOV Alexander S. , ZARETSKAYA Malvina Sh. , YAROTSKY Sergei V. , NAUMOVA Irina B. , CHIZHOV Oleg S. , SHABAROVA Zoye A.
Published: March 2005

The cell walls of Streptomyces antibioticus 39 contain a glycosylated poly(glycero1 phosphate), in which the repeating monomeric unit is O‐α‐d‐galactopyranosyl‐(3–3)‐O‐2‐acetamido‐2‐deoxy‐β‐d‐galactopyranosyl‐(1–1)‐glycerol monophosphate.

The localization of the phosphodiesters linkages between hydroxyl groups at positions 2 and 3 of the adjacent glycerol residues and the structure of the glycoside were established by 13C nuclear‐magnetic‐resonance spectroscopy. The spectral data are also in accordance with the results of the methylation analysis and enzyme degradation of the glycoside.
The Binding of 8‐Anilinonaphthalene‐1‐sulphonate to a Fixed Concentration of Unenergised and Succinate‐Energised Submitochondrial Particles
Authors: GAINS Nigel , DAWSON Alan P.
Published: March 2005

The effect of the approximately hyperbolic relationship between fluorochrome concentration and light absorbed on the interpretation of data for the binding of 8‐anilinonaphthalene‐1‐sulphonate to unenergised and succinate‐energised submitochondrial particles has been investigated. If this is taken into account plots of fluorescence against fluorescence × [fluorochrome]−1 do not tend towards a maximum fluorescence value. The significance of this findings is discussed.
Premature Appearance of Hepatic Phosphoenolpyruvate Carboxykinase in Fetal Rats, Not Mediated by Adenosine 3′: 5′‐Monophosphate
Authors: MENCHER David , SHOUVAL Daniel , RESHEF Lea
Published: March 2005

Injection of streptozotocin in utero to fetuses elicited a premature appearance of cytosolic hepatic activity of phosphoenol pyruvate carboxykinase. This was due to a precocious initiation of the synthesis of the enzyme. The streptozotocin‐induced appearance of enzyme activity was not mediated by adenosine 3′:5′‐monophosphate since the concentration of the cyclic nucleotide in the liver was unaffected by the antibiotic, the administration of dibutyryladenosine 3′:5′‐monophosphate to streptozotocin‐treated fetuses elicited an additive increase in enzyme activity, and insulin administration in utero repressed the streptozotocin effect while the effect due to dibutyryladenosine 3′:5′‐monophosphate was not inhibited by simultaneous insulin injection.

Streptozotocin treatment also caused a small but consistent retardation of fetal growth and a marked reduction of liver wet weight. Histological analysis of the liver demonstrated a premature loss of some hematopoietic elements, while hepatocytes appeared normal. Hepatic protein synthesis was unaffected by the streptozotocin treatment.

Streptozotocin treatment had no effect on fetal renal phosphoenol pyruvate carboxykinase activity or kidney wet weight.
An Anti‐(Signal Peptide) Antibody: Purification, Properties and Use as a Conformational Probe
Authors: BATY Daniel , LAZDUNSKI Claude
Published: March 2005

An antibody was raised against the precursor form of alkaline phosphatase. The antiserum was depleted of the immunoglobulin G (IgG) directed against the mature alkaline phosphatase. A pure anti‐(signal peptide) antibody was then obtained by affinity chromatography using a column of Sepharose linked to the signal peptide by the C‐terminal portion. As expected from the size of the antigen, this antibody did not precipitate with the peptide suggesting that this peptide contains only one antigenic determinant. Thus, a double immunoprecipitation system, with an anti‐rabbit IgG raised in goat, was used. In this system with the mature alkaline phosphatase very little precipitation occurred, showing that the antibody isolated was mostly directed against the signal peptide. Not only the signal peptide from the precursor form of the alkaline phosphatase but also that from the precursor form of another periplasmic protein: the leucyl‐isoleucyl‐valine binding protein, was recognized by the antibody. This suggests that conformational homologies exist between the signal peptides of precursors of periplasmic proteins. Aminopeptidase N, a protein located on the outer face of the cytoplasmic membrane, was precipitated by the anti‐(signal peptide) antibody in the double immunoprecipitation system. This peripheral membrane protein could also inhibit the precipitation of the signal peptide. Because the signal sequences usually show no consistent homology, we propose that a conformation of signal peptides is recognized by the antibody isolated. This hypothesis is discussed.
A Method for the Estimation of Esterase Synthesis and Degradation and its Application to Evaluate the Influence of Insulin and Glucagon
Authors: HEYMANN Eberhard , MENTLEIN Rolf , SCHMALZ Rainer , SCHWABE Christian , WAGENMANN Frank
Published: March 2005

The irreversible reaction between liver esterases and the active‐site‐directed inhibitor bis(4‐nitrophenyl)phosphate can be used in vivo both for the estimation of the esterase contents and for the measurement of the esterase degradation rates. A method based on this reaction is described which allows the simultaneous estimation of the rate constants of degradation and synthesis of esterases during a period of change in protein concentration. Rat liver was found to contain about 1 mg of organophosphate‐binding esterases per g of fresh tissue while the microsomal fraction contains about 30 mg of esterases per g of microsomal protein. Esterase degradation and de novo synthesis were shown to remain in equilibrium for a period of at least five days following the injection of 10 mg bis(4‐nitro‐[14C]phenyl)phosphate per kg. The decrease of the relative amount of labeled esterases with time was found to follow first‐order kinetics yielding an average esterase degrading constant of 0.0165 h−1 which corresponds to a half‐life of 42 h. These data were confirmed by an independent experiment using one of the standard procedures for the estimation of degradation rates: [14C]leucine was incorporated and one of the esterases was subsequently isolated by immunoprecipitation. Using isoelectric focussing and dodecyl sulfate electrophoretic methods, the various esterase isoenzymes appeared to have very similar, if not identical turnover rates.

This method for the estimation of the turnover characteristics was applied to evaluate hormone effects on liver esterases. The time course of the contents and the turnover of liver esterases was measured under the influence of glucagon treatment in diabetic rats and under the influence of high doses of insulin. The esterase content decreased faster than the average content of microsomal protein under the influence of glucagon. The reverse effect was observed with insulin‐treated rats. Both insulin and glucagon apparently reduced the intracellular esterase turnover in rat liver. Kinetic analysis of the results revealed that insulin mainly lowered the esterase degradation rate, though the rate of esterase synthesis might also have been restricted. In the glucagon‐treated rats the de novo synthesis of esterases was strongly reduced.