bluuebunny/crossref_metadata_2025 huggingface dataset:
A Contribution of Nonhistone Proteins to the Conformation of Chromatin
Authors: TASHIRO Tomoko , KUROKAWA Masanori
Published: March 2005

Changes in circular dichroism (CD) spectra and thermal melting profiles of guinea pig liver DNA reassociated with histones and/or nonhistone proteins from the cerebral or liver chromatin are described.

In the DNA · histone complex, positive ellipticity in the CD spectrum at 260–300 nm is progressively lowered as the histone/DNA ratio is increased; this is accompanied by a red‐shift of the crossover point at around 260 nm. DNA in this complex is thermally stabilised to a considerable extent, but not to such a full extent as is shown with DNA in native chromatin.

DNA · nonhistone complex in 0.14 M NaCl is, in contrast to DNA · histone complex, not precipitable by centrifugation at 20000 × g. DNA in this complex shows only a slight reduction in ellipticity at 260–300 nm, and a very weak thermal stabilisation.

Characteristics in the CD spectrum of the native chromatin are most satisfactorily reproduced in the DNA · histone · nonhistone complex. These include a large decrease in ellipticity at 260–300 nm, a red‐shift of the crossover point at around 260 nm, and a slight negative band at around 305 nm. Also, DNA in this complex is thermally stabilised to the extent comparable with DNA in the native chromatin.

Addition of nonhistone proteins to the preformed DNA · histone complex in 3 M urea renders a half of the complex, named DNA · histone(· nonhistone), unprecipitable upon centrifugation at 20000 ×g in 0.14 M NaCl. CD spectrum and thermal melting profile of the precipitable DNA · histone(·nonhistone) complex are similar to those of the DNA · histone · nonhistone complex, while in the unprecipitable DNA · histone(· nonhistone) complex, the ellipticity at 260 – 300 nm is significantly elevated and the highest melting transition (at 80 °C) is lacking.

The CD spectrum of native cerebral chromatin closely resembles that of unprecipitable DNA · histone(·nonhistone) complex, while in liver chromatin, the spectrum is an intermediate between those of the unprecipitable and precipitable complexes, suggesting an organ‐specific modification of chromatin by nonhistone proteins. Cerebral nonhistone proteins bind to DNA and to the DNA · histone complex more extensively than liver nonhistone proteins.

It is concluded that, although the basic conformation of DNA in native chromatin is determined largely by histones, nonhistone proteins also play an individual role. There is also an indication that nonhistone proteins exert an organ‐specific modification of chromatin superstructure.
Structure and Synthesis of a Lipid‐Containing Bacteriophage
Authors: TSUKAGOSHI Norihiro , PETERSEN Marianne H. , FRANKLIN Richard M.
Published: March 2005

Enzymes of glycerophosphatide biosynthesis and degradation have been characterized using membrane fractions of Pseudomonas BAL‐31, the host cell of the lipid‐containing bacteriophage PM2. Inner and outer membrane fractions were investigated. Whereas the outer membrane con‐tained more phosphatidylethanolamine and less phosphatidylglycerol than the inner membrane, the fatty acid composition was almost the same for both membranes. The pathway for glycerophos‐phatide biosynthesis was the same as that for Escherichia coli and Bacillus megaterium. The biosyn‐thesis of phosphatidylserine and phosphatidylglycerol was shown to proceed via CDP‐diglyceride as a common intermediate. Phospholipase A hydrolyzed phosphatidylethanolamine more efficiently than phosphatidylglycerol. All of the enzymatic activities were stimulated by Triton X‐100. Phos‐phatidylserine synthetase was stimulated by Na2SO4 and inhibited by Mg2+. Phosphatidylserine decarboxylase was completely inhibited by NH2OH. Phosphatidylglyceror synthetase was stimulated by Mg2+. Phospholipase A was stimulated by Ca2+. Phosphatidylserine decarboxylase and phosphatidylglycerol synthetase were localized in the inner membrane fraction. Phosphatidylserine synthetase and phospholipase A were found in both outer and inner membrane fractions.

Effects of virus infection on these enzymes were studied. Phosphatidylglycerol synthetase and phospholipase A activity against phosphatidylethanolamine were activated early post‐infection. Phosphatidylserine synthetase and decarboxylase remained unaltered. At later times post‐infection, phosphatidylglycerol synthetase and phosphatidylserine decarboxylase were inactivated and phos‐ phatidylserine synthetase was activated. There was little alteration in phospholipase A activity against phosphatidylglycerol after infection.
Characterization of Calf‐Ovary Adenosine‐3′: 5′‐Monophosphate‐Dependent Protein Kinases and Adenosine‐3′: 5′‐Monophosphate‐Binding Proteins
Authors: TALMADGE Kenneth W. , BECHTEL Elke , SALONKANGAS Anneli , HUBER Peter , JUNGMANN Richard A. , Eppenberger Urs
Published: March 2005

Protein phosphokinase activity from the calf ovary cytosol (105000 × g supernatant fraction) has been resolved by chromatography and polyacrylamide gel electrophoresis into two major pro‐tein kinases, PK‐H1 and PK‐H2, both dependent on adenosine 3′: 5′‐monophosphate (cyclic AMP). The enzymes have similar molecular weights (230000) and substrate specificities but differ in their cyclic‐AMP‐dependency and stimulation by cyclic AMP. The differences have been explained by the presence in PK‐H1 of a unique cyclic‐AMP‐binding protein which has little catalytic activity as‐ sociated with it. The cyclic‐AMP‐binding protein has a high affinity for cyclic AMP and in addition is able to inhibit the activity of the isolated catalytic subunit.

The ovarian cyclic‐AMP‐dependent protein kinases have properties similar to those found in other tissues. They can be dissociated into catalytic and regulatory subunits and are inhibited by a heat‐stable protein inhibitor isolated from rabbit skeletal muscle.

Preincubation of the cytosol with high levels of cyclic AMP resulted in additional cyclic‐AMP‐dependent protein kinases and cyclic‐AMP‐binding proteins which include protein kinases and binding proteins of > 400000 molecular weight.
The Peptide Components of Bee Venom
Authors: GAULDIE Jack , HANSON Jennifer M. , RUMJANEK Franklin D. , SHIPOLINI Rudolf A. , VERNON Charles A.
Published: March 2005

A procedure is described for the simultaneous separation of all the peptides (other than those present in trace amounts) contained in the venom of the common European honey bee (Apis mellifera). Three new peptides have been isolated: one is a fragment of the known peptide melittin. Some properties of the peptides are described.
Circular‐Dichroism and Absorption Spectroscopic Studies on Specific Aromatic Residues Involved in the Different Modes of Aggregation of Tobacco‐Mosaic‐Virus Protein
Authors: VOGEL Dieter , JAENICKE Rainer
Published: March 2005

Conformational changes accompanying the different modes of aggregation of tobacco mosaic virus protein (TMV‐protein) were investigated using circular dichroism (CD) and absorption difference spectra in the range of aromatic absorption. Comparing wild‐type protein and mutant Ni 2068 (Tyr‐139→Cys‐139) a tentative localization of aromatic amino acids in the threedimensional structure is rendered possible.

In all modes of aggregation the CD spectra are determined by intrasubunit interactions between aromatic residues, in particular Trp‐17 and Trp‐52 as well as Tyr‐70, Tyr‐72 and Tyr‐139. The Trp‐1 7Trp‐52 interaction was found to be highly sensitive towards changes of the quaternary structure especially with respect to helical aggregates. This suggests that the environment of the two tryptophan residues is of crucial importance in the three‐dimensional structure of the subunit; in the course of aggregation intersubunit interactions compete with the specific intrasubunit Trp‐17‐Trp‐52 interactions.

It is suggested that Tyr‐70 and Tyr‐72 form hydrogen bonds in a strongly hydrophobic environment. Formation of the double disc decreases the rotatory strength, pointing to an increase in conformational flexibility.

Spectroscopic and chemical evidence prove that Tyr‐70, Tyr‐72 and Tyr‐139 are in close neighbourhood. Double disc formation by lowering the pH (pH 8 → 6.9, I = 0.1 M) or increasing the ionic strength (pH 8, I = 0.1 → 0.6 M) is reflected by identical spectral effects in the environment of Tyr‐70‐Tyr‐72. However the interaction between Trp‐17 and Trp‐52 indicates significant differences in the conformation which may be important for the formation of higher aggregates, i.e. ‘lockwashers’, helices, and ‘stacked discs’.
Cortisol Modification of HeLa 65 Alkaline Phosphatase
Authors: BAZZELL Kirstan L. , PRICE Gary , TU Sylvia , GRIFFIN Martin , COX Rody , GHOSH Nimai
Published: March 2005

Alkaline phosphatase activity of HeLa cells is increased 5–20‐fold during growth in medium with cortisol. The increase in enzyme activity is due to an enhanced catalytic efficiency rather than an increase in alkaline phosphatase protein in induced cells. In the present study the chemical composition of control and induced forms of alkaline phosphatase were investigated to determine the enzyme modification that may be responsible for the increased catalytic activity. HeLa alkaline phosphatase is a phosphoprotein and the induced form of the enzyme has approximately one‐half of the phosphate residues associated with control enzyme. The decrease in phosphate residues of the enzyme apparently alters its catalytic activity. Other chemical components of purified alkaline phosphatase from control and induced cells are similar; these include sialic acid, hexosamine and sulfhydryl residues.
The Protein‐Mediated Transfer of Phosphatidylcholine between Membranes
Authors: WIRTZ Karel W. A. , GEURTS VAN KESSEL Wouter S. M. , KAMP Hein H. , DEMEL Rudolf A.
Published: March 2005

The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine‐containing vesicles; zwitterionic phosphatidylethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg 2+and, to a lesser extent, K+ overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K+ affected the membrane surface charge relatively little inhibition was observed. In measuring the protein‐mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order Las3+ < Mg2+≤ Ca2+≤ K+= Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine
Chemical Modification as a Probe of the Topography and Reactivity of Horse‐Spleen Apoferritin
Authors: WETZ Klaus , CRICHTON Robert R.
Published: March 2005

In apoferritin, but not in ferritin, 1.0 ± 0.1 cysteine residue per subunit can be modified. In ferritin 3.3 ± 0.3 lysine residues and 7.1 ± 0.7 carboxyl groups per subunit can be modified, whilst the corresponding values for apoferritin are 4.4 ± 0.4 lysine residues and 11.0 ± 0.4 carboxyl groups per subunit. Modification of lysine residues with maleic anhydride and carboxyl groups with glycineamide in apoferritin which has been dissociated and denatured in guanidine hydrochloride leads to the introduction of 9.1 ± 0.5 maleyl groups per subunit and 22.0 ± 0.9 glycineamide residues per subunit. Whereas unmodified apoferritin subunit can be reassociated from guanidine hydrochloride to apoferritin monomer, the ability of maleylated apoferritin to reassociate is impaired. Apoferriitin in which all the carboxyl groups have been blocked with glycineamide cannot be reassociated to apoferritin and exists in solution as stable subunits.

The modification of one cysteine residue per subunit, of 3 or 4 lysine residues per subunit or of 7 carboxyl groups per subunit has no effect on the catalytic activity of apoferritin. In contrast the modification of 11 carboxyl groups per subunit completely abolishes the catalytic properties of the protein. We conclude that one or more carboxyl groups are essential for the catalytic activity of horse spleen apoferritin.
The Structure of Teichoic Acid from Bacillus subtilis var. niger WM as Determined by 13C Nuclear‐Magnetic‐Resonance Spectroscopy
Authors: DE BOER Willem R. , KRUYSSEN Fred J. , WOUTERS Jan T. M. , KRUK Cornelis
Published: March 2005

The walls of Bacillus subtilis var. niger WM, grown in a Mg2+ ‐limited chemostat culture (carbon source glucose, dilution rate = 0.2 h−1, 37 °C, pH 7) contained 45 % (w/w) teichoic acid, a polymer composed of glycerol, phosphate and glucose in the molar ratio 1.00: 1.00: 0.88, respectively.

Alkaline hydrolysis of this teichoic acid yielded 1‐O‐β‐glucosylglycerol phosphate (together with small amounts of glycerol phosphate) and 13C nuclear magnetic resonance spectra of this hydrolysis product, and its derivative after alkaline phosphatase treatment, confirmed, that the monomeric unit was 1‐O‐β‐glucosylglycerol‐3‐phosphate. Assignment of the resonances in the spectrum of undegraded teichoic acid revealed that the polymer was a poly[(2,3)glycerol phosphate], glucosidically substituted on C‐1 of glycerol with β‐glucose.
A Proton‐Relaxation‐Time Study of the Conformation of Some Purine and Pyrimidine 5′‐Nucleotides in Aqueous Solution
Authors: CHACHATY Claude , ZEMB Thomas , LANGLET Gerard , SON TRAN DINH , BUC Henri , MORANGE Michel
Published: March 2005

The measurement of proton relaxation time T1 in a series of purine and pyrimidine 5′‐nucleotides has been carried out to investigate their conformation in dilute neutral aqueous solutions.

The interpretation of relaxation data has been performed with the help of computer calculations taking into account the different conformers of the ribose ring and of the exocyclic group. It has been found that all nucleotides under study show nearly the same preferential orientations of the base defined by r0= 70°± 10° in the syn range. A more elaborate treatment, using an angular distribution derived from calculated potentials on 5′‐GMP gives theoretical relaxation times in close agreement with experimental ones.
Anion and Amine Uptake and Uncoupling in Submitochondrial Particles
Authors: AZZONE Giovanni Felice , GUTWENIGER Heidi , VIOLA Elena , STRINNA Ermanno , MASSARI Stefano , COLONNA Raffacle , Pregnolato L.
Published: March 2005

Unlike chloroplasts, submitochondrial particles are not uncoupled by nigericin + KCl or NH4Cl. Also the uncoupling effect of lipophilic anions is largely independent of the addition of weak bases.

Low concentrations of permeant anions cause a shift of the steady‐state energy level rather than a cycle of energy utilization. The degree of inhibition of ATP synthesis by tetraphenylboron is larger than required for the uptake of the anion.

Lipophilic anions such as bromthymolblue, bromcresolpurple, and 8‐anilino‐1‐napthalene sulphonate cause a pH‐independent, 50% uncoupling in submitochondrial particles at concentrations of 3, 30 and 30 μM, respectively. The passive interaction of bromthymolblue and bromcresol‐purple appears as a pH‐dependent distribution between two phases. ATP causes a pH‐independent slight shift in the anion distribution, with negligible anion accumulation.

Addition of amines to energized submitochondrial particles results in two types of effects: uptake of amines and uncoupling. While in chloroplasts amine uptake and uncoupling are closely associated, this is not the case in submitochondrial particles. The uncoupling effect is observed only with lipophilic and not with hydrophilic amines, and the degree of uncoupling increases with the lipophilicity of the amines. The amine uptake, on the other hand, is accompanied by negligible uncoupling.

While the uptake of amines is dependent on the presence of non‐permeant anions, such as C1−the uncoupling effect is independent of Cl−. Furthermore the amine uncoupling is markedly enhanced by lipophilic anions.

The view is discussed that the uncoupling effect of lipophilic anions and lipophilic amines in submitochondrial particles is due to a catalytic energy dissipation rather than to a stoichiometry energy utilization. The molecular mechanism of uncoupling presumably involves a cycling of charges after a perturbation of the membrane structure.
Kinetics of Adenosine 3′:5′‐monophosphate Accumulation in Dog Thyroid Slices
Authors: SWILLENS Stéphane , VAN SANDE Jacqueline , POCHET Roland , DELBEKE Dominique , PICCART Martine , PAIVA Manuel , DUMONT Jacques E.
Published: March 2005

Dog thyroid slices have been stimulated in vitro by thyrotropin. The kinetics of adenosine 3′:5′‐monophosphate (cyclic AMP) accumulation due to the activation of adenylate cyclase were measured. Experimental results have been interpreted by means of a numerical simulation based on a theoretical description of the overall process which consists of three steps: (a) the penetration of thyrotropin in the slices, (b) the binding of the hormone to the specific receptor and the consequent activation of adenylate cyclase, (c) the kinetics of cyclic AMP accumulation in each cell due to the interplay between synthesis and degradation. The numerical values of the parameters needed for the simulation were deduced from separate experiments. Diffusion of thyrotropin in the slices has been calculated from the kinetics of efflux of [3H]sucrose, and with or without albumin. Adenylate cyclase activity and activation by thyrotropin in homogenates and purified membranes were measured. Binding experiments of cyclic AMP on crude extract of dog thyroid lead to the conclusion that the maximal capacity of the specific binding site is close to the cyclic AMP content in resting thyroid cells. The purpose of this paper is to verify the validity of the current concepts about the mechanisms taking place in this process by comparing experimental results and theoretical simulation. It results from the study that for this system the time between the activation of adenylate cyclase and half maximal cyclic AMPaccumulation is equal to 3 min 45 s and the pool of cyclic AMP is renewed three times per minute. It has been also stressed that the thickness of the slices as well as inhibitors of phosphodiesterases change the apparent kinetics of cyclic AMP accumulation.
Effect of Triiodothyronine on Rat Liver Chromatin Protein Kinase
Authors: KRUH Jacques , TICHONICKY Lydie
Published: March 2005

Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin non‐histone proteins. A significant increase was found after two daily injections. A 4‐fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p‐hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3,3′,5′‐triiodothyronine have no effect on protein kinase.

Chromatin non‐histone proteins isolated from rats injected with triiodothyronine incorporated more 32P when incubated with [γ‐32P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32P incorporation.

It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non‐histone proteins.
The Involvement of Protein L 11 in the Joining of the 30‐S Initiation Complex to the 50‐S Subunit
Authors: NAAKTGEBOREN Nico , SCHRIER Peter , MÖLLER Wim , VOORMA Harry O.
Published: March 2005

Ribosomal protein L11 participates in the coupling of the 30‐S initiation complex with the 50‐S subunit.

P37 cores, lacking L7, L8, L12, L33, L10 and L11 were reconstituted with L7 and L10. These particles are unable to join successfully to the 30‐S initiation complex, whereas reconstitution of the same cores in the presence of L7, L10 and L11 restores 60–80% of the original coupling activity. P0 cores lacking only L7, L8, L12 and L33 are able to carry out one round of initiation, addition of L7 resulting in complete restoration of full activity. The data obtained with these P37 core particles resemble those obtained with untreated 50‐S particles carrying thiostrepton, which prevents the binding of initiation factor IF‐2 into the 70‐S initiation complex.

It is postulated that L11 induces a niche on the ribosomal surface to facilitate the proper binding of the IF‐2 . GTP fMet‐tRNA complex. This binding of IF‐2 enables the 30‐S initiation complex to join to the 50‐S subunit, because of the associative ability of IF‐2. If joining is impaired then both the level of fMet‐tRNA binding and of the IF‐2‐mediated GTP hydrolysis is lowered.
Purification and Properties of Formaldehyde Dehydrogenase and Formate Dehydrogenase from Candida boidinii
Authors: SCHÜTTE Horst , FLOSSDORF Josef , SAHM Hermann , KULA Maria‐Regina
Published: March 2005

Formaldehyde hydrogenase and formate dehydrogenase were purified 130‐fold and 19‐fold respectively from Candida boidinii grown an methanol. The final enzyme preparations were homogeneous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weights of the enzymes were determined by sedimentation equilibrium studies and calculated as 80000 and 74000 respectively. Dissociation into subunits was observed by treatment with sodium dodecylsulfate. The molecular weights of the polypeptide chains were estimated to be 40000 and 36000 respectively.

The NAD‐linked formaldehyde dehydrogenase specifically requires reduced glutathione for activity. Besides formaldehyde only methylglyoxal served as a substrate but no other aldehyde tested. The Km values were found to be 0.25 mM for formaldehyde, 1.2 mM for methylglyoxal, 0.09 mM for NAD and 0.13 mM for glutathione. Evidence is presented which demonstrates that the reaction product of the formaldehyde‐dehydrogenase‐catalyzed oxidation of formaldehyde is Sformvlglutathione rather than formate. The NAD‐linked formate dehydrogenase catalyzes specifically the oxidation of formate to carbon dioxide. The Km values were found tobe 13 mM for formate and 0.09 mM for NAD.
Renaturation of Bacteriophage Lambda DNA
Authors: FANNING Thomas G. , SCHREIER Peter H. , DAVIES R. Wayne
Published: March 2005

Reannealed hybrid molecules of wild‐type bacteriophage lambda DNA were prepared in aqueous solutions of formamide at a variety of NaC1 concentrations at both room temperature (∼ 22 °C) and 37 °C. Treatment of the hybrid DNA molecules with the single‐strand‐specific nuclease S1 from Aspergillus oryzae followed by alkaline sucrose gradient sedimentation was used to monitör the extent and fidelity of hybridization.

The optimal renaturation conditions at room temperature were found to be: 50% formamide, 35–55 mM NaCl and 10 mM Tris‐HCI (pH 8.5) at 20–25 μg DNA/ml. Optimal conditions at 37 °C were:32% formamide, 35–55 mM NaCl and 10 mM Tris‐HC1 (pH 8.5) at 20–25μg DNA/ml. Under these conditions approximately 85–90 % of the input single‐stranded DNA (molecular weight 1.5 × 107) was rendered S1‐nuclease‐resistant within 8 h at room temperature and 5 h at 37 °C. Neither Mg2+ nor spermidine appeared to have an effect an either the extent or fidelity of duplex formation.

Experiments performed with excess enzyme and with lambda/lambda imm 434 heteroduplex hybrids suggested that the hybrid DNA molecules formed under optimal conditions contained no, or only short (<1%), mismatched regions.
The Reconstitution of Anacystis nidulans DNA‐Dependent RNA Polymerase from Its Isolated Subunits
Authors: HERZFELD Frank , KIPER Manuel
Published: March 2005

The DNA‐dependent RNA polymerase of the blue‐green alga Anacystis nidulans was reconstituted from its isolated subunits in the absence of urea. Applying this technique the kinetics and the subunit requirements of the reconstitution process were analyzed. The results reveal differences with respect to the reconstitution of Escherichia coli polymerase.

Reconstitution proceeds much more slowly in the case of the A. nidulans enzyme. Reconstitution here is absolutely dependent an the presence of the subunit σ. On the other hand, the largest of the subunits of M, = 190000 can be fully substituted by a specific degradation product of this subunit of Mr= 175000.

Heterologous reconstitution between subunits of E. coli and A. nidulans polymerase does not result in active enzyme hybrids, showing a divergent evolution of the structure of this enzyme in these procaryotic organisms.
Yeast Ribosomal Proteins are Synthesized on Small Polysomes
Authors: MAGER Willem H. , PLANTA Rudi J.
Published: March 2005

Yeast polysomes were fractionated into various size classes and then the nascent polypeptide chains an each separate class of polysomes were elongated in vitro. Analysis of the completed polypeptides by polyacrylamide gel electrophoresis demonstrated that the polysomes engaged in ribosomal protein synthesis Kave a relatively small size
A Plasmocytoma Ribosome‐Associated Protein Kinase which Phosphorylates a Specific Protein of the Ribosomal KCl Wash
Authors: QUIRIN‐STRICKER Christiane , SCHMITT Michéle , EGLY Jean‐Marc , KEMPF Jules
Published: March 2005

One ribosomal protein kinase activity and three soluble protein kinase activities have been identified in plasma cell tumors by DEAE‐cellulose chromatography. We have shown phosphorylation in vivo and in vitro of a protein fraction from the ribosomal KCl wash which we have termed ‘PPx fraction’. Phosphorylation of this protein fraction has been obtained in vitro with the ribosomeassociated protein kinase. We have determined for the ribosomal protein kinase the following characteristics.

It is an Mg2+‐dependent enzyme that transfers the γ‐phosphate from ATP into phosphoseryl and phosphothreonyl residues of the substrate.

It has a wide substrate specificity. Like the soluble protein kinases it catalyses the phosphorylation of several proteins like histone, phosvitin, casein and ribosomal proteins but it differs from the main soluble kinases (I, II) by the fact that it catalyses specifically the phosphorylation at least of one of the ribosomal KCl wash proteins. On dodecylsulfate‐polyacrylamide gels this protein has a molecular weight of approximately 90000 and it is released from ribosomes under conditions commonly employed for extraction of initiation factors.

The ribosome‐associated protein kinase is not stimulated by the addition of cyclic adenosine 3′: 5′‐monophosphate.

KCl has no effect, NaCl has a weak effect on.the phosphorylation, Mn2+ and Ca2+ are inhibitors.

ADP has been found to be a competitive inhibitor:

The maximum velocity of the ribosomal protein‐kinase‐catalysed reaction is 0.65 nmol of 32P incorporated in the KCI wash protein per min and per mg protein.

The apparent Km for the ribosomal KCl wash protein äs substrate is 0.71 mg/ml and the Km for ATP is 94 μM.

The molecular weight of the ribosomal protein kinase, estimated by electrophoresis in polyacrylamide‐dodecylsulfate gels, is 60000 and corresponds probably to a catalytic subunit.
Evidence that DNA Polymerase‐a of Calf Thymus Contains a Subunit of Molecular Weight 155000
Authors: HOLMES Andrew M. , HESSLEWOOD Ian P. , JOHNSTON Irving R.
Published: March 2005

Small samples of the 8‐S species of enzymes (A1 and A2) occurring in the DNA polymerase‐a fraction of calf thymus, have been extensively purified using non‐denaturing (normal) polyacrylamide gel electrophoresis. When peak fractions of activity on normal gels were subjected to dodecylsulphate‐polyacrylamide gel electrophoresis, a polypeptide at 155000 correlated with polymerase activity. Samples of the 7.3‐S (C) enzyme prepared from A2 by treatment with 2.4 M urea or isolated directly without exposure to urea, also showed the presence of a 155000‐Mr polypeptide. It is concluded that the 7.3‐S (C) enzyme, of previously estimated molecular weight 155000–170000, is a single polypeptide and that the 8‐S enzymes A1 and A2 contain an additional subunit of 50000–70000 molecular weight.
Enzyme‐Linked Immunoassay
Authors: KATO Kanefusa , HAMAGUCHI Yoshitaka , FUKUI Hideo , ISHIKAWA Eiji
Published: March 2005

Rabbit immunoglobulin G (IgG) was reduced by incubating with 10 mM 2‐mercaptoethylamine and then treated with excess amounts of N,N′‐O‐phenylenedimaleimide. As a result, maleimide residues were introduced into rabbit IgG molecules. Rabbit IgG containing maleimide residues could be coupled to β‐d‐galactosidase from Escherichia coli which has sulfhydryl groups in the molecule. The resulting rabbit IgG (antibody)–enzyme complex may be useful for immunoassay of antigens. As an example, human IgG was assayed by the sandwich method using the rabbit anti‐(human IgG) IgG‐β‐d‐galactosidase complex and amounts of human IgG as small as 3 fmoles were measurable.
Organization of Vitellogenin Polysomes, Size of the mRNA and Polyadenylate Fragment
Authors: JOST Jean‐Pierre , PEHLING Gundula
Published: March 2005

A heavy polysome fraction containing vitellogenin mRNA was isolated from the liver of oestradiol‐treated chicks. As determined by urea – polyacrylamide gel electrophoresis, the molecular weight of vitellogenin mRNA is about 2.5 × 106. The mRNA contains a polyadenylate segment of about 220 nucleotides at the 3′ end. The remaining 7000 nucleotides are sufficient to code for a polypeptide of Mr about 270000. Combining ‘run off’ experiments of heavy polysomes in vitro together with radioimmunoprecipitation and polyacrylamide gel electrophoresis of the translation product, we concluded that vitellogenin mRNA is probably monocistronic and the 2.5 × 106−Mr mRNA codes for two polypeptides, Mr 30000 and 240000. The largest polypeptide is, in our cellfree system and liver homogenate, readily cleaved into smaller peptides.
Accumulation of Replicative DNA Intermediates in Tetrahymena after Excision‐Repairable Damage to DNA
Authors: JOHNSON Brian , WESTERGAARD Ole
Published: March 2005

Inhibition of DNA synthesis in the eukaryotic organism Tetrahymena pyriformis by thymine starvation results in the formation and accumulation of a distinct class of DNA during the first hour after re‐initiation of the synthesis. This DNA, when synthesized in the presence of 5‐bromodeoxyuridine (BrdUrd), bands as a distinct peak at an intermediate density of Q = 1.693 g/cm3 in a neutral CsCl buoyant density gradient. Following short pulses of BrdUrd, the DNA of intermediate density accounts for up to 90% of the newly synthesized DNA and binding assays on nitrocellulose filters show the presence of single‐stranded regions. Alkaline buoyant density and alkaline velocity gradients clearly demonstrate the presence of non‐covalently linked newly synthesized fragments with an average length shorter than that of the parental strands. Pulse experiments show that the DNA of intermediate density is progressively converted to fully hybrid (light · heavy) DNA molecules. It is therefore suggested that in response to damage caused by thymine starvation, a replicative DNA intermediate accumulates at the growing point. A similar phenomenon has also been observed after irridation with ultraviolet light. The effect of the protein synthesis inhibitor cycloheximide on the formation on the intermediate and the fully hybrid material is also described.
Norepinephrine‐Induced Shift in Levels of Adenosine 3′:5′‐monophosphate and ATP Parallel to Increased Respiratory Rate and Lipolysis in Isolated Hamster Brown‐Fat Cells
Authors: PETTERSSON Bertil , VALLIN Ivar
Published: March 2005

Isolated brown fat cells from hamster respond to added catecholamines with a temporary increase in respiratory rate and an extended lipolysis.

From experiments with catecholamines and α and β‐blockers, the receptors of these cells are classified as β according to classical definition.

Norepinephrine induces a rapid increase in adenosine 3′:5′‐monophosphate levels which parallels in time the stimulated respiration. Maximal adenosine 3′:5′‐monophosphate levels are reached within 1–3 min and are followed by a continuous decline.

Parallel to the catecholamine‐induced respiration and lipolysis there is a pronounced drop in ATP levels. This energy depletion could be reversed by addition within 5 min after norepinephrine of the β‐blocker propranolol.

The nucleotide pattern in isolated hamster brown fat cells after norepinephrine addition was mimicked in experiments with isolated hamster brown fat mitochondria. From these experiments it is concluded that a high ratio of AMP and ADP over ATP decreases the respiratory rate when endogenous free fatty acids are oxidized.
The Enzymic Addition of Poly(A) to the 3′‐End of RNA using Bacteriophage MS 2 RNA as a Model System
Authors: DEVOS René , GILLIS Elie , FIERS Walter
Published: March 2005

ATP: RNA adenyltransferase, purified from Escherichia coli, was used to add a series of adenosine residues to the 3′‐end of MS2 RNA. Incubations of the order of a few minutes at 37 °C were sufficient for synthesis of a short poly(A) chain that did not appreciably alter the hydrodynamic or electrophoretic properties of MS2 RNA. The size of the poly(A) tails was estimated by gel electrophoresis after prior hydrolysis of the primer RNA with pancreatic ribonuclease. These results were in good agreement with the values calculated on the basis of the relative amount of incorporated AMP. After the addition of a short poly(A) tail, approximately 50 % of the treated material binds specifically to an oligo(dT)‐cellulose column. The majority of the recovered poly(A)‐containing RNA was still intact, as shown by analysis on polyacrylamide gel.

After incubations beyond 6 min, slowly sedimenting material, also showing reduced electrophoretic mobility, was formed. Presumably this material corresponds to RNA chains to which long poly(A) tails are linked.
Comparison of Cytoplasmic and Nuclear Poly(A)‐Containing RNA Sequences in Xenopus Liver Cells
Authors: RYFFEL Gerhart U.
Published: March 2005

Poly(A)‐containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared.

After denaturation of the RNA by dimethylsulfoxide the average molecule of nuclear poly(A)containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)‐containing RNA sediments slightly ahead of 18 S.

To compare the complexity of cytoplasmic and nuclear poly(A)‐containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)‐containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)containing RNA. An attempt to isolate a specific probe for this nucleus‐restricted poly(A)‐containing RNA reveals that more than 104 different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasts. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.
Molecular Modifications Associated with Ageing of Globin Messenger RNA in vivo
Authors: NOKIN Pierre , HUEZ Georges , MARBAIX Gérard , BURNY Arsène , CHANTRENNE Hubert
Published: March 2005

Using polyacrylamide gel elution‐electrophoresis in aqueous medium, highly purified rabbit globin mRNA can be. fractionated into several populations of molecules differing by their mean poly(A) content. Both α and β globin mRNA are heterogeneous with respect to their electrophoretic mobilities. With the conditions used no separation of α and β globin mRNA occurs during electrophoresis. From the specific radioactivity distribution in the different mRNA fractions one can conclude that the polyadenylate sequence at the 3′ end of globin mRNA molecules becomes shorter with ageing. This shortening occurs on α, as well as β, globin mRNAs and the extent of heterogeneity in poly(A) content is similar for both globin mRNAs. Furthermore, using two different methods of mRNA fractionation (polyacrylamide gel elution‐electrophoresis and elution of poly(U)‐Sepharose‐bound mRNA at increasing temperatures) it is shown that old mRNA molecules differ from relatively young messages in their ability to direct cell‐free globin synthesis. Modifications reducing template activity in vitro thus seem to take place during globin mRNA ageing
Homoserine Kinase from Escherichia coli K12
Authors: BURR Benjamin , WALKER John , TRUFFA‐BACHI Paolo , COHEN Georges N.
Published: March 2005

Homoserine kinase was purified to apparent homogeneity from a derepressed strain of Escherichia coli K12, using standard fractionation techniques. It is a dimer (Mr= 60000) composed of apparently identical polypeptide chains (Mr= 29 000). Its amino acid composition and N‐terminal sequence have been determined. l‐Threonine is a competitive inhibitor of the substrate l‐homoserine; this inhibition is straightforward and shows no sign of co‐operativity. Evidence is presented that homoserine and threotine bind to the same site of this non‐allosteric enzyme. The binding of homoserine and threonine can also be studied by difference spectroscopy; the latter studies reveal an unexpected effect of magnesium ions, which might be the basis for the unusual high Mg2+ requirement for optimal enzyme reaction
A Quantitative Model for Partition in Aqueous Multiphase Systems
Authors: JOHANSSON Göte , HARTMAN Annika
Published: March 2005

A model for the partition of charged molecules in aqueous multiphase systems has been developed. The partition coefficient of one component, or the overall partition coefficient of a number of components, between two arbitrary phases is expressed in terms of the difference in electrical potential between the phases (due to electrolytes present in the system), the net charges of the partitioned components and their partition coefficients in a (sometimes hypothetical) uncharged state. The fraction of material in one phase has also been described as a function of the net charges of the partitioned components.

The model fits well to experimental data for partition of chromate, pyridine, ribonuclease A, two types of CO‐hemoglobin and an enzyme mixture (yeast lysate) in three‐phase systems consisting of poly(ethylene glycol), dextran, Ficoll and water. Minor deviations from the model are construed to be a pH‐dependent uptake of ions. The data have also been used to detect differences in solvation of similar proteins, as well as the presence of several forms of some glycolytic enzymes present in yeast lysate.
Spin‐Labelled AMP –an Activator of Phosphorylase
Authors: BUSBY Stephen J. W. , HEMMINGA Marcus A. , RADDA George K. , TROMMER Wolfgang E. , WENZEL Herbert
Published: March 2005

A spin‐labelled AMP derivative and its diamagnetic analogue activate phosphorylase b in the same way, but do not activate phosphorylase a.

The electron‐spin‐resonance spectra of the spin‐labelled AMP derivative bound to phosphorylase b and a have ‘powderlike’ characteristics indicating that the spin label is immobilised on the protein. From changes in the electron‐spin‐resonance spectrum of spin‐labelled AMP as phosphorylase b or a is added, the dissociation constants were calculated.

The interactions of spin‐labelled AMP and the diamagnetic analogue with phosphorylase b and a have been monitored by observing changes in the spectral properties of fluorescent and spinlabel probes covalently attached to the enzyme.

The dissociation constants of spin‐labelled AMP and phosphorylase b or a are 175 ± 25 μM and 15 ± 5 M respectively. Similar dissociation constants are obtained for the diamagnetic analogue. The effect of these AMP derivatives on the covalently attached probe groups and on phosphorylase activity is compared to the effect of AMP and IMP.
Equilibrium Studies on the Formaldehyde Reaction with Native DNA
Authors: SHIKAMA Keiji , MIURA Ken‐Ichi
Published: March 2005

Equilibrium analysis of the reaction of formaldehyde with native calf thymus DNA was carried out at temperatures below the thermal transition zone by the spectrophotometric method. The apparent equilibrium constant, Kconf., for the conformational opening and closing reaction of base pairs along the double‐helical chain, was measured in various concentrations of formaldehyde, and these values were extrapolated to the zero concentration. The value of K0conf. thus obtained in the absence of the chemical probe was 0.12 in 5 mM 2‐(N‐morpholino)ethane sulfonic acid buffer (Mes), and 0.003 in 0.15 M NaCl plus 5 mM Mes, pH 7.0, at 50 °C. These results make it possible to calculate roughly that 230 base pairs and 7 base pairs are open respectively, at 50 °C in the native DNA molecule composed of 2300 base pairs, using Mr= 1.5 106 for the sample used.

This conformational reaction was also characterized by the following thermodynamic parameters: ΔG0conf= 1.36 kcal · mol−1 (5.68 kJ · mol−1)ΔH0conf.= 36.8 kcal · mol−1 (154 Kj ·mol−1), and ΔSconf= 110 cal·mol−1·K−1 (460 J · mol−1· K−1) in 5 mM Mes, pH 7.0, at 50 °C, and the nature of the ‘breathing’ of base pairs was discussed.
Transcription from Complementary Deoxyribonucleic Acid Strands in Various Sporogenic and Asporogenic Mutants of Bacillus subtilis
Authors: BONAMY Céline , MANCA de NADRA Maria‐Cristina , SZULMAJSTER Jekisiel
Published: March 2005

The present paper describes an investigation, at the transcription level, in a thermosensitive sporulation mutant of Bacillus subtilis (ts‐4) grown at the permissive (30 °C) or restrictive (42 °C) temperature where sporulation capacity is respectively expressed or arrested. These studies were carried out by analysing the ribonucleic acid from vegetative and stationary phase cells (t3 cells) grown under both conditions, by hybridization‐competition experiments.

It was found that about 32 % of t3 RNA species synthesized on the heavy (H) strand and about 19% of those synthesized on the light (L) strand have distinct sequences, depending on the growth conditions of the ts‐4 cells: permissive or restrictive conditions. No such difference was observed in a normal zero‐stage asporogenic mutant grown under the same conditions.

These results suggest, therefore, that at the restrictive temperature the block in sporulation capacity of this mutant might, at least partially, reside in a deficiency at the transcription level.

The significance of transcription from the L strand of B. subtilis DNA reported by others was also investigated. Based on our hybridization‐competition studies on different sporogenic and asporogenic mutants it was suggested that the expression of some vegetative genes on the L strand during sporulation might have a regulatory function in the ‘switch‐on’ of spore genes on the H stand.
The Use of a Cleavable Crosslinking Reagent to Identify Neighboring Proteins in the 30‐S Ribosomal Subunit of Escherichia coli
Authors: PERETZ Hava , TOWBIN Harry , ELSON David
Published: March 2005

A cleavable bifunctional reagent, dimethyl 3,3′‐dithiobispropionimidate, has been used to crosslink proteins that occupy neighboring positions in the 30‐S ribosomal subunit of Escherichia coli. The crosslinked proteins were identified, fully or partly, by their positions in two twodimensional gel electrophoretic systems, one diagonal and the other quasi‐diagonal, in which the complexes were cleaved alter the first‐dimensional run. It was found to be necessary to block the protein sulfhydryl groups in order to prevent artifactual disulfide crosslinking after extraction of the protein from the ribosome. Eleven crosslinked complexes were detected. Four were fully identified: the triplet S4‐S5‐S8, and the pairs S2‐S3, S4‐S5, and S5‐S8. In five others one component was identified unambiguously. No additional complexes were seen when the longer homologous butyro and capro reagents were used
Methylation Analysis of Glucosaminitol and Glucosaminyl‐glucosaminitol Disaccharides
Authors: HASE Sumihiro , RIETSCHEL Ernst Th.
Published: March 2005

N‐Acetylglucosaminitol, N‐acetylglucosaminyl‐β 1′,4‐N‐acetylglucosaminitol (chitobiitol), the corresponding trimer (chitotriitol) and N‐acetylglucosaminyl‐β 1′,6‐N‐acetylglucosaminitol were subjected to methylation analysis using combined gas‐liquid chromatography/mass spectrometry. The preparations were subjected to permethylation according to Hakomori, followed by acid hydrolysis. reduction and peracetylation. From N‐acetylglucosaminitol and its 4‐0 and 6‐0 substituted derivatives, methylated N‐acetylacetamido derivatives were obtained. The possible conditions for the formation of these products are discussed.
Isolation and Analysis of the Lipid A Backbone
Authors: HASE Sumihiro , RIETSCHEL Ernst Th.
Published: March 2005

A degradation procedure of lipopolysaccharides was worked out which allows the isolation of the reduced backbone of lipid A in a total yield of between 20 and 30 %. This procedure was applied to lipopolysaccharides of S forms (Salmonella minnesota, Shigella flexneri 5 b, Escherichia coli 086, E. coli 0111, Xanthomonas sinensis, Rhodopseudomonas gelatinosa) and R mutants (Salmonella minnesota, Shigella flexneri 5b, E. coli BB9 and E. coli EH 100). Chemical analysis, reaction with β‐N‐acetyl‐glucosaminidase and application of methylation analysis revealed that the lipid A backbone of all strains contains β 1′,6‐linked glucosamine disaccharides carrying two phosphate groups, one in glycosidic and one in ester linkage, a structure, identified previously in the Salmonella minnesota Re mutant.