bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence of a Specifically Encapsidated RNA Fragment Originating from the Tobacco‐Mosaic‐Virus Coat‐Protein Cistron
Authors: GUILLEY Hubert , JONARD Gérard , RICHARDS Kenneth Eugene , HIRTH Léon
Published: March 2005

When 25‐S tobacco mosaic virus (TMV) Protein aggregate and TMV RNA, which has been partially digested by T1 RNase, are mixed under conditions suitable for reconstitution, only a few RNA fragments are encapsidated. These fragments were isolated and purified by polyacrylamide gel electrophoresis. The sequence of the three main fragments, the longest of which (fragment 1) was estimated to contain 103 nucleotides, has been determined. The two smaller fragments are portions of the longer chain produced by an additional specific scission. Because of the great affinity of 25‐S TMV protein for this nucleotide sequence, it will be referred to as the “specifically encapsidated RNA fragment”. The occurrence of a “hidden break” in the sequence has been demonstrated: fragment 1, purified by electrophoresis on a polyacrylamide gel without 8 M urea, gives rise upon further electrophoresis in the presence of urea to two new bands corresponding to the two halves of the molecule. A stable hair‐pin secondary structure has been derived from the base sequence which can account for the specificity of action of the enzyme. Because of its properties, we have suggested elsewhere that the sequence of fragment 1 might correspond to the disk recognition site for reconstitution, which is known to be located at the 5′ end of the intact RNA. But experiments with TMV RNA whose 5′ ‐OH end has been radioactively phosphorylated with polynucleotide kinase show that this is not the case. Analysis of the amino acid coding capacity of the fragment has instead revealed that fragment 1 is a portion of the TMV coat protein cistron.
Two Enzymically Active Forms of Glycyl‐tRNA Synthetase from Bacillus brevis
Authors: SURGUCHOV Andrei P. , SURGUCHOVA Irina G.
Published: March 2005

Using sucrose density centrifugation and gel filtration of a 105000 ×g supernatant of Bacillus brevis two enzymic activities of glycyl‐tRNA synthetase were separated. Enzyme catalyzing the aminoacylation of tRNA (E1) elutes in a high‐molecular‐weight region. Enzyme active in glycylhydroxamate formation (E2) elutes from a Sephadex gel column and sediments in sucrose density gradient in a region of relatively low molecular weight. The presence of two enzymic activities does not depend on the method of cell disruption; their proportion does not change when protease inhibitor (diisopropylphosphorofluoridate) is added to the extraction buffer.

Both E1 and E2 were purified to a nearly homogeneous state. Sedimentation coefficients (Sw.20) were found to be 8.6 S and 3.6 S and molecular weights 226000 and 66000 for E1 and E2, respectively. During storage, E1 dissociates into two components, one of which has electrophoretic mobility identical to E2. The molecular weight of the other component is about 160000.

Electrophoresis of E1 in the presence of sodium dodecylsulfate reveals two bands corresponding to molecular weights of 81000 and 30000. Under these conditions, E2 dissociates into a polypeptide with a molecular weight of 30000.

Valine was found to be the N‐terminal amino acid for E2 and both valine and glutamic acid were N‐terminal amino acids for E1.

It is concluded that E1 is a tetrameric protein consisting of two large and two small subunits (α2β2). E2 is a component of E1 with a structural formula α2.
Proton‐Magnetic‐Resonance Studies of the Lysine Residues of Ribonuclease A
Authors: BROWN Larry R. , BRADBURY J. Howard
Published: March 2005

The amino groups of ribonuclease A (RNase‐A) have been methylated with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance (PMR) studies. Although enzymatic activity is lost, PMR difference spectroscopy and PMR studies of thermal denaturation show native conformation is largely preserved in methylated RNase‐A. Resonances corresponding to the NH2‐terminal α‐amino and 10 ɛ‐amino N‐methyl groups are titrated at 220 MHz to obtain pK values. After correction for the effects of methylation, using values previously derived from model compound studies, a pK of 6.6 is found for the α‐amino group, a pK of 8.6 for the ɛ‐amino group of lysine‐41 and pK values ranging from 10.6 to 11.2 for the other lysine ɛ‐amino groups. Interactions between lysine‐7 and lysine‐41 or between the α‐amino and ɛ‐amino groups of lysine‐1 have been proposed to account for deviations from simple titration behaviour.

The correct continuities for the titration curves of the histidine H‐2 proton resonances have been confirmed by selective deuteration of the H‐2 protons. Titration curves for the H‐2 proton resonances of histidine‐12 and histidine‐119 of methylated RNase‐A show deviations from the titration curves for the native enzyme, indicating some alteration of the active‐site conformation. In the presence of phosphate, titration curves for the H‐2 proton resonances of histidine‐12 and histidine‐119 of methylated RNase‐A indicate binding of phosphate at the active site, but these curves continue to show deviations from the titration behaviour of native RNase‐A. The titration curve for the N‐methyl resonance of lysine‐41 is perturbed considerably by the presence of phosphate, which indicates a possible catalytic role for lysine‐41.
The Isolation of Nuclei and Basic Nucleoproteins from the Cellular Slime Mold Dictyostelium discoideum
Authors: CHARLESWORTH Marilyn Chapleo , PARISH Roger W.
Published: March 2005

A method is described for the isolation of nuclei from an axenic strain of Dictyostelium discoideum using a sorbitol/Ficoll solution and low concentration of Triton X‐100.

Basic proteins have been extracted from the nuclei and on polyacrylamide gel electrophoresis yield a consistent pattern in which five major groups or bands predominate. Four of these five fractions comigrate with calf thymus histones and one fraction seems to be unique to D. discoideum.

The slowest moving of the five fractions is soluble in 0.5 M perchloric acid and comigrates with calf thymus histone F1. After recovery from the perchloric acid solution by precipitation with acetone this fraction yielded one major band on electrophoresis.
Translational Step Inhibited in vivo by Aflatoxin B1 in Rat‐Liver Polysomes
Authors: SARASIN Alain , MOULÉ Yvonne
Published: March 2005

Aflatoxin B1 strongly inhibits protein synthesis in rat liver cells. We previously demonstrated that this inhibition could be divided into two steps: up to 5h aflatoxin blocks protein synthesis directly and specifically at the polysome level; beyond 7h protein synthesis inhibition appears chiefly as a consequence of transcription impairment due to drug action.

This paper confirms the foregoing results and represents an attempt to localize the translational step inhibited in vivo by aflatoxin B1. We used the simulation study developed by Li, Kisilevsky, Wasan and Hammond, 1972 (Biochim. Biophys. Acta, 272, 451–462) to determine precisely the site inhibited in vivo after drug intoxication. This analysis is based on two parameters: the kinetics of polysome labeling to follow the nascent peptide synthesis, and the kinetics of supernatant labeling to follow the completed protein synthesis.

Up to 5h after dosing, aflatoxin specifically inhibits the elongation and/or termination steps during protein synthesis; after longer periods of time inhibition occurs essentially at the initiation step. When the intracellular concentration of aflatoxin is too high, particularly 2h after dosing, each step of protein synthesis is blocked.

Polypeptide synthesis by the postmitochondrial supernatants isolated from aflatoxin‐treated animals is impaired in the same proportion as protein synthesis in vivo. The damage caused by aflatoxin is mostly observed on microsomes. However, purified polysomes isolated from aflatoxin‐treated rats synthesize proteins in vitro to the same extent as those from controls. These results suggest that aflatoxin metabolite(s) are bound to polysomes with noncovalent bonds. These active metabolites are probably lost during polysome isolation procedures.

Finally, relationships between protein metabolism and aflatoxin carcinogenesis are discussed.
Covalent Attachment of DNA to Agarose
Authors: ARNDT‐JOVIN Donna J. , JOVIN Thomas M. , BÄHR Wolfgang , MARQUARDT Maria , FRISCHAUF Anne‐Marie
Published: March 2005

DNA has been covalently linked to insoluble matrices of agarose (Sepharose) in high yield using cyanogen bromide activation. Both double‐stranded and single‐stranded DNA have been coupled with yields up to 225 nmol/mg dry weight Sepharose or 3–8 μmol nucleotide phosphate/ml bed volume. The DNA‐Sepharose has been used for (a) the affinity chromatography of various enzymes (Escherichia coli DNA polymerase I and RNA polymerase) from crude extracts or after initial purification steps, resulting in high yields and degrees of purification, and for (b) nucleic acid hybridization. The DNA‐Sepharose is stable to high temperature, prolonged storage, and in the case of single‐stranded DNA, can be washed with NaOH to destroy nuclease activity and to release any digested oligonucleotides or mononucleotides.
Interaction of a Non‐Histone Chromatin Protein (High‐Mobility Group Protein 2) with DNA
Authors: GOODWIN Graham H. , SHOOTER Kenneth V. , JOHNS Ernest W.
Published: March 2005

The interaction with DNA of the calf thymus chromatin non‐histone protein termed the high‐mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125I‐labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263–270] for the interaction of high‐mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high‐mobility group protein 2 differs from high‐mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form.

The molecular weight of high‐mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26000 was obtained.

A low level of nuclease activity detected in one preparation of high‐mobility group protein 2 has been investigated.
Sterol Biosynthesis in Yeast
Authors: BOLL Meinrad , LÖWEL Marianne , STILL Jaroslava , BERNDT Jürgen
Published: March 2005

Anaerobically and aerobically grown yeast contains 3‐hydroxy‐3‐methylglutaryl‐CoA reductase, which is located in the mitochondrial fraction of the cell. Anaerobically grown yeast has a low specific activity of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase and a low sterol content. Aeration of this yeast in buffer, without growth, results in an increase in the specific activity of the enzyme, which is paralleled by an increase in the sterol content. This induction has an oscillatory profile with yeast grown anaerobically for 24 h and a linear pattern with cells grown anaerobically for 72 h. With the latter type of yeast, glucose is necessary for an induction, whereas with the other yeast an induction occurs with and without glucose. By an anaerobic incubation in buffer of the yeast grown anaerobically for 24 h, the oscillatory profile can be transformed into a linear one. The extent of induction of the reductase is strictly dependent on the concentration of glucose present. Sterols increase in whole cells, but they do not increase in the mitochondrial fraction. The induction of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase is strongly inhibited by cycloheximide, but is not affected by chloramphenicol. The induction of the enzyme is closely connected with the glucose metabolism of the cell; fructose, mannose, and ethanol can also induce the reductase.
Spin‐Labeling Studies of Urea‐Treated Leucine Aminopeptidase
Authors: LASSMANN Günter , DAMERAU Werner , SKLENAR Gisela , SCHWARZ Dieter , FROHNE Marlies , LUDEWIG Manfred , HANSON Horst
Published: March 2005

Leucine aminopeptidase (EC 3.4.11.1) from bovine eye lens was spin‐labeled at the most reactive thiol groups with 2,2,6,6‐tetramethyl‐4‐[2‐iodoacetamido]‐piperidine‐1‐oxyl.

Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A:B ∼ 4:1 was estimated. In B a hydrophobic environment of label was observed.

Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes.

a) An additional weakly immobilizing conformational state (B′) with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B′ shows a time‐dependent increase of its extent at the expense of B and A′ (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn2+.

b) Mn2+ complexed with the protein is partly released producing hydrated Mn2+.

c)After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A:B ∼ 4:1 even after long incubation.

6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely.
Mechanism of 2‐Deoxy‐d‐glucose Inhibition of Cell‐Wall Polysaccharide and Glycoprotein Biosyntheses in Saccharomyces cerevisiae
Authors: KRÁTKÝ Zdeněk , BIELY Peter , BAUER Štefan
Published: March 2005

The mechanism of inhibition by 2‐deoxy‐d‐glucose of the synthesis of yeast wall polysaccharides and glycoproteins was investigated in Saccharomyces cerevisiae cells and protoplasts. The extent of the inhibition of mannan and glucan synthesis was found to be dependent on whether glucose or mannose was used as the carbon source in the medium. During growth on glucose, 2‐deoxy‐d‐glucose inhibited more intensively mannan than glucan formation. Biosynthesis of wall glucan was strongly suppressed in mannose medium. Selective incorporation of 2‐deoxy‐d‐glucose occurred into that polysaccharide, synthesis of which was more inhibited under given conditions. Suggestive evidence has been obtained that the decisive factor for the proportion of glucan and mannan in the walls is the direction of glucose 6‐phosphate/mannose 6‐phosphate interconversion dependent on the exogeneous hexose.

No close correlation was found between the inhibition of mannan synthesis and the appearance of the mannan‐protein enzymes invertase and acid phosphatase. Effect of 2‐deoxy‐d‐glucose was therefore investigated on the parallel synthesis of protein, mannan and several extracellular and intracellular enzymes in protoplasts grown on glucose and mannose. The results obtained pointed out that the hindrance of the secretion of mannan‐protein enzymes is of a complex nature and related more to the inhibition of synthesis of the protein moiety than to the inhibition of glycosylation. Synthesis of several enzymes was found to be a subject of a metabolic control by 2‐deoxy‐d‐glucose or its metabolites.
Modification of Amino Groups of Human‐Erythrocyte Glycoproteins and the New Concept on the Structural Basis of M and N Blood‐Group Specificity
Authors: LISOWSKA Elwira , DUK Maria
Published: March 2005

Various kinds of modification of amino groups of M and N blood group glycoproteins abolished their capacity to inhibit rabbit and human anti‐M and anti‐N sera.

The reversible modification of amino groups revealed that M and N blood group activity was restored after the removal of amino‐group‐blocking residues.

Modification of amino groups had an entirely different effect on the reactivity of red cell glycoproteins with Vicia graminea agglutinin. The serological activity of N glycoprotein towards Vicia graminea anti‐N agglutinin was unchanged, whereas the weak activity of M glycoprotein towards this anti‐N agglutinin was increased to the level of that of N glycoprotein.

These results indicate that there is a structural difference between M and N glycoproteins, which resides beyond the oligosaccharide chains. It suggests in turn that M and N blood group specificity is determined by amino acid sequence in the peptide chains of red cell glycoproteins.
Effects of Ricin on the Ribosomal Sites Involved in the Interaction of the Elongation Factors
Authors: CARRASCO Luis , FERNANDEZ‐PUENTES Carmen , VAZQUEZ David
Published: March 2005

The effects of ricin on the different steps of the elongation cycle of protein synthesis in a rabbit reticulocyte cell‐free system are studied in this paper. The toxin most probably acts by catalytically inactivating the ribosomes, since a single molecule of the toxin can inactivate 300 ribosomes for poly(U)‐directed phenylalanine incorporation. The effect of the toxin on the ribosome is irreversible.

Ricin specifically inhibits elongation‐factor‐1‐dependent aminoacyl‐tRNA binding to ribosomes but has no effect on the non‐enzymic binding of aminoacyl‐tRNA. Ricin also inhibits formation of the complex elongation‐factor‐2 · ribosome · nucleotide with GTP, GDP or GMP‐P(CH2)P. However, the toxin has no effect on translocation. These apparently conflicting results are discussed in this study.
Trans‐N‐Deoxyribosylase: Purification by Affinity Chromatography and Characterization
Authors: HOLGUIN José , CARDINAUD Robert
Published: March 2005

trans‐N‐Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans‐N‐deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans‐N‐deoxyribosylase‐I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans‐N‐deoxyribosylase‐II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine‐specific enzyme is a polymeric enzyme of molecular weight 86000 (± 4000).
Trans‐N‐Deoxyribosylase: Substrate Specificity Studies
Authors: HOLGUIN José , CARDINAUD Robert , SALEMINK C. A.
Published: March 2005

A series of purine bases and analogues were tested as substrates for trans‐N‐deoxyribosylase (EC 2.4.2.6). It was observed that the pyrimidine ring and its substituents on positions 1, 2 and 6, are of minor importance. On the other hand only a few modifications are tolerated on the imidazole moiety, as follows.

A tautomeric proton must be present on the imidazole ring. The “usual” shift is between position 9 and 7.

The position of the tautomeric proton governs the site of substiution.

For steric reasons no substituent is allowed on position 8.
On the Interaction of Esters and Peptides with Carboxypeptidase B
Authors: ZISAPEL Nava , SOKOLOVSKY Mordechai
Published: March 2005

The specificity of porcine carboxypeptidase B towards basic and non‐basic substrates was studied by employing several esters of phenyllactate. The structure of these depsipeptides complement exactly those of the corresponding phenylalanyl oligopeptide substrates. These non‐basic ester‐peptide pairs as well as the basic ester‐peptide pair of arginyl derivatives, permits the direct comparison of the pH dependencies of the kinetic constants for the hydrolysis of those substrates by carboxy‐peptidase B. The data is interpreted in terms of three specific ionizing groups located at the active site of the enzyme. The mode and extent of inhibition of the hydrolysis of a specific substrate by another substrate was characterized kinetically. These results are discussed in relation to a proposed model for esterolytic and proteolytic action of carboxypeptidase B.
Lipophilic Proteins Encoded by Mitochondrial and Nuclear Genes in Neurospora crassa
Authors: KÜNTZEL Hans , PIENIA̧ŻZEK Norman J. , PIENIA̧ŹEK Danuta , LEISTER Dennis E.
Published: March 2005

Mitochondrial proteins soluble in neutral chloroform—methanol (2:1) were separated from lipids by ether precipitation and resolved by Sephadex G‐200 filtration in the presence of dodecylsulfate into two major fractions eluting in the excluded region (peak I) and in a region of an apparent molecular weight 8000 (peak II). Residual phospholipids are found only in peak II. Peak I consists of several aggregated small polypeptides of molecular weights around 8000, which can be disaggregated by mild oxidation with performic acid.

Cycloheximide stimulates almost two‐fold incorporation of radioactive phenylalanine into peak I proteins but inhibits labelling of peak II proteins by 95%. Chloramphenicol and ethidium bromide inhibit the synthesis of peak I proteins by 70% and 95% respectively, but do not affect labelling of peak II proteins.

At least 30% of the translation products of mitochondrial DNA in vitro behave like peak I proteins: they are soluble in organic solvents, they aggregate in dodecylsulfate buffer after removal of phospholipids and they contain species of molecular weights around 8000 that disaggregate upon oxidation.

The data strongly suggest that the proteins of peak I are encoded by mitochondrial genes and synthesized on mitochondrial ribosomes, whereas the proteins of peak II are encoded by nuclear genes and synthesized on cytoplasmic ribosomes.

Both groups of lipophilic proteins are very similar in their molecular weights, but the mitochondrially coded peak I proteins have the unique property of forming large heat‐stable aggregates in the presence of dodecylsulfate.
The Involvement of Sulphydryl Groups in the Peptidyl Transferase Centre of Eukaryotic Ribosomes
Authors: CARRASCO Luis , VAZQUEZ David
Published: March 2005

Treatment of mammalian ribosomes with N‐ethylmaleimide enhances up to 100% the ribosome efficiency in the “fragment reaction assay” for peptide bond formation by increasing the affinity of the substrate C‐A‐C‐C‐A‐Leu‐Ac for the donor site. This stimulation in peptidyl transferase activity was not observed when yeast ribosomes were treated in a similar manner.

Stimulation of the peptidyl transferase activity of mammalian ribosomes was also observed by treatment with either p‐chloromercuribenzoic acid or 5,5′‐dithiobis‐(2‐nitrobenzoic acid) or 5.5′‐dithiobis‐(2‐nitropyridine) or the maleimide‐derived antibiotic showdomycin.

N‐Ethylmaleimide treatment also enhances C‐A‐C‐C‐A‐Leu binding to the acceptor site of the peptidyl transferase centre. However, neither binding of N‐Ac‐Phe‐tRNA in the presence of ethanol, nor binding of Phe‐tRNA to the ribosomes is stimulated by N‐ethylmaleimide.

The antibiotic tenuazionic acid (a selective inhibitor of peptide bond formation by mammalian ribosomes) appears to require for its inhibitory effect the ribosome sulphydryl residues, since its inhibitory action on the fragment reaction is greatly decreased in ribosomes treated with N‐ethylmaleimide.
Metabolism of trans‐3‐Hexadecenoic Acid in Broad Bean
Authors: HARWOOD John L. , JAMES Anthony T.
Published: March 2005

Broad bean (Vicia faba) leaves contain rather high concentrations (about 4% of total fatty acids) of the trans‐3‐hexadecenoic acid.

Amounts of the acid increase with the age of the leaves and are absent from etiolated tissue.

Changes in the levels of trans‐Δ3‐hexadecenoic acid can be produced by subjecting the intact plants to various light/dark periods.

Chloroplasts isolated from broad‐bean leaves show high rates of fatty acid synthesis from [1‐14C]acetate. Synthesis is dependent on coenzyme A and ATP but is insensitive to the addition of exogenous acyl carrier protein.

The pattern of acids made includes about 20% palmitic, 5% hexadecenoic, 10% stearic and 60% oleic. trans‐3‐Hexadecenoic acid synthesis was most active in chloroplasts from plants exposed to the dark for 5 days and light for 3 days.

Arsenite addition inhibited stearate formation by isolated chloroplasts but resulted in a two‐fold stimulation of overall synthesis.

The rate of fatty acid synthesis by isolated chloroplasts paralleled the changes in endogenous trans‐3‐hexadecenoic acid levels in the leaves from which they were isolated.
ATP‐Sensitive and ATP‐Insensitive Phosphofructokinase in Escherichia coli K‐12
Authors: DOELLE Horst W.
Published: March 2005

The purification and kinetic characteristics of two phosphofructokinases are described. Aerobic cultures of Escherichia coli exhibit two types of phosphofructokinase. Both types are dimers of mol. wt 150000 (subunit mol. wt 73000), whereas the anaerobic culture of E. coli revealed only one type, which is a tetramer of mol. wt 350000 (subunit mol. wt 90000). Type 1 of the aerobic enzyme, representing approximately 70% of the total enzyme activity, is ATP‐insensitive, whereas type II and the anaerobic enzyme are ATP‐sensitive.

The addition of AMP stimulates the tetramer, relieving ATP inhibition, and also the type II dimer, which is, however, inhibited at concentrations higher than 0.5 mM AMP. No effect was observed on the type I dimer of the aerobic preparation. ADP stimulates the tetramer and inhibits type I more strongly than type II of the aerobic dimer.

The kinetic characteristics together with the effect of metabolites on these phosphofructokinase types are described and discussed in the light of their importance for the regulatory mechanism of the Pasteur effect.
Properties of ATP and UTP Analogues with P‐S‐C5′ Bonds
Authors: STÜTZ Anton , SCHEIT Karl Heinz
Published: March 2005

Analogues of ATP and UTP bearing C5′ ‐S‐P ester bonds were found not to be substrates but weak competitive inhibitors of Escherichia coli RNA polymerase. The Ki values of the analogues obtained in the transcription of poly[d(A‐T)] or poly(dT) under various conditions are in the order of millimolar. Evidence was derived from proton magnetic resonance spectra that nucleotides with C5′ ‐S‐P bonds do not exist in gauche‐gauche conformation normally adopted by natural occurring nucleotides. This leads us to assume that the gauche‐gauche conformation is an essential prerequisite for substrates of RNA polymerase. Ado‐5′‐S‐PPP substituted for ATP as substrate of hexokinase from yeast rather effectively thus indicating that a distinct stereochemical orientation of the α‐phosphate ester bond is not a stringent requirement for substrates of this enzyme.
Flavin‐Nicotinamide Biscoenzymes: Models for the Interaction between NADH (NADPH) and Flavin in Flavoenzymes
Authors: BLANKENHORN Gunter
Published: March 2005

Flavin‐nicotinamide biscoenzymes covalently linked by two, three or four methylene groups through positions N(10) of the flavin (F1) and N(1) of the nicotinamide (Nic) form long‐wavelength‐absorbing, intramolecular complexes when the flavin part of the molecule is reduced specifically. The energy of the long‐wavelength transition is minimal and its intensity maximal for Nic+ox‐(CH2)3‐FlredH−(λmax= 600 nm; ɛ= 1200 M−1× cm−1).

The increasing proximity of the positively charged nicotinamide lowers the pK‐value of dihydroflavin deprotonation up to 1.7 units and the flavin oxidation‐reduction potential becomes more positive up to 116 mV.

Specific reduction of the nicotinamide part of the biscoenzymes yields transient, long‐wavelength‐absorbing complexes. The energy of the long‐wavelength transition is minimal and its intensity maximal for the complex NicredH‐(CH2)3‐Flox(λmax= 575 nm; ɛ= 650 M−1× cm−1).

The rate of intramolecular flavin‐dependent dihydronicotinamide dehydrogenation is highest for NicredH‐(CH2)3‐Flox(345 s−1), about 3 times slower for NicredH‐(CH2)4‐Floxand 100 times slower for NicredH‐(CH2)2‐Flox.

The results obtained in this study are consistent with a reaction mechanism that involves formation of a charge transfer complex between reduced nicotinamide and oxidized flavin and rate‐limiting heterolytic breakdown into products.
Conformational Studies of Equilibrium Structures in Fragments of Horse Heart Cytochrome c
Authors: TONIOLO Claudio , FONTANA Angelo , SCOFFONE Ernesto
Published: March 2005

Ultraviolet absorption and circular dichroism studies have been carried out on horse heart apo‐cytochrome c and heme‐free peptide fragments obtained by cyanogen bromide cleavage of the native protein. It was noted that the various peptides assume predominantly an unordered conformation in water solution. Increasing ionic strength and addition of 2‐chloroethanol increase the right‐handed helical content. Guanidinium hydrochloride favors the coil state. It was also demonstrated that two non‐interacting helical regions of different stability are present in the apo‐protein in 2‐chloroethanol.
A Pulse Fluorometry Study of Lipoamide Dehydrogenase
Authors: WAHL Philippe , AUCHET Jean‐Claude , VISSER Antonie J. W. G. , VEEGER Cees
Published: March 2005

The time dependence of the fluorescence of tryptophanyl and flavin residues in lipoamide dehydrogenase has been investigated with single‐photon decay spectroscopy.

When the two FAD molecules in the enzyme were directly excited the decay could only be analyzed in a sum of two exponentials with equal amplitudes. Ths phenomenon was observed at 4°C (τ1= 0.8 ns, τ2= 4.7 ns) and at 20 °C (τ1= 0.8 ns, τ2= 3.4 ns) irrespective of the emission and excitation wavelengths. This result reveals a difference in the nature of the two FAD centers.

By excitation at 290 nm the fluorescence decay curves of tryptophan and FAD were obtained. The decays are analyzed in terms of energy transfer from tryptophanyl to flavin residues. The results, which are in good agreement with those obtained previously with static fluorescence methods. show that one of the two tryptophanyl residues within the subunit transfers its excitation energy to the flavin located at a distance of 1.5 nm.
Determination of the Kinetic Constants of Glucose‐6‐phosphate 1‐Epimerase by Non‐Linear Optimization
Authors: CHANCE Edwin M. , HESS Benno , PLESSER Theodor , WURSTER Bernd
Published: March 2005

The overall kinetic constants of the reversible anomerisation of d‐glucopyranose 6‐phosphate from α to β non‐enzymatically as well as catalysed by glucose‐6‐phosphate 1‐epimerase are determined by application of a novel computerized non‐linear optimization technique.

The non‐enzymic rate constants for the anomerisation of d‐glucopyranose 6‐phosphate from α to β and reverse are 0.0658 and 0.0389 s−1, respectively. The Michaelis constants of the enzymic reaction are Kαm 144 μM and Kβm 55.5 μM with the turnover numbers of 1950 s−1 and 446 s−1 for the conversion of d‐glucopyranose 6‐phosphate from α to β and reverse, respectively.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K12
Authors: HIRTH Christian G. , VÉRON Michel , VILLAR‐PALASI Carlos , HURION Nicole , COHEN Georges N.
Published: March 2005

2‐Amino‐4‐oxo‐5‐chloropentanoic acid inactivates specifically the homoserine dehydrogenase activity of the bifunctional enzyme, aspartokinase I‐homoserine dehydrogenase I. The aspartokinase activity remains essentially untouched and retains its threonine sensitivity. The inactivation of the dehydrogenase requires the covalent binding of one equivalent of the analogue per subunit. Alkylation does not affect the tetrameric state of the protein. The alkylating agent, a substrate analogue, meets the qualitative and quantitative requirements of an affinity label.
Procollagen Biosynthesis by Embryonic‐Chick‐Bone Polysolnes
Authors: VUUST Jens
Published: March 2005

Both total polysomes and polysomes of different size classes isolated from embryonic chick cranial bones were allowed to complete their nascent polypeptide chains in a cell‐free system containing rabbit reticulocyte post‐ribosomal supernatant fraction. In this system, no de novo initiation of polypeptide synthesis occurred. The product was analysed for relative content of proαl and proα2, the precursors of the α chains of collagen, by dodecylsulphate‐acrylamide gel electrophoresis as, well as by paper electrophoresis following tryptic digestion. The results showed that the products of polysome protein synthesis contained proαl and proα2 in the 2:1 ratio in which the corresponding a chains are present in native collagen, and that proαl and proα2 synthesising polysomes are of the same size. These findings, in conjunction with results from a previous report [Vuust, J. & Piez, K. A. (1972) J. Biol. Chem. 247, 856–862] suggest that active messenger ribonucleic acids for the proαl and proα2 chains, respectively, are present in the cells in a ratio of 2: 1, and that the rates of initiation, elongation, termination and release from nolvsomes are all identical for the two chains.
Rapid Isolation of Highly Active RNA Polymerase from Escherichia coli and Its Subunits by Matrix‐Bound Heparin
Authors: STERNBACH Hans , ENGELHARDT Reinhild , LEZIUS A. G.
Published: March 2005

RNA polymerase from Escherichia coli is selectively and strongly retained by a heparin‐substituted agarose and can be eluted therefrom by a neutral buffer containing 0.6 M salt. The method is applicable to relatively crude preparations of the enzyme on a preparative scale giving highly purified RNA polymerase in excellent yield. The enzyme obtained by this procedure shows the highest specific activity so far reported and is pure and enriched in factor σ as indicated by dodecylsulfate gel electrophoresis.

Based on the differential affinity of the subunits of the enzyme for the heparin‐carrying gel matrix, a method for separation of α, β' +β and σ subunits by application of urea and salt‐containing buffers is described. Upon recombination and dialysis with urea‐free buffer 40–50% of the enzyme activity is restored.
Intermolecular Renaturation of Hairpin Loops Leads to Stable and Specific Aggregates of 28‐S rRNA and Precursor rRNAs from Xenopus laevis
Authors: HAGENBÜCHLE Otto , SCHIBLER Ueli , WYLER Toni
Published: March 2005

Under certain extraction conditions, 28‐S rRNA of Xenopus laevis can form specific dimers by intermolecular renaturation of a particular hairpin loop near the B end of the molecules. This was demonstrated by electron microscopy of isolated aggregates under conditions where rRNA shows a reproducible pattern of secondary structure. In 50% formamide, 15 mM sodium chloride, 1.5 mM trisodium citrate, pH 7.0, the aggregates dissociate into their monomers at a temperature. (61 0C) at which the most stable hairpin loops of the 28‐S rRNA are melting. Precursor rRNAs containing the 28‐S rRNA region also form aggregates with 28‐S rRNA by the same linkage mechanism. Since such artificial aggregates yield molecular weights higher than expected for true precursors of Xenopus rRNA, they may interfere in precursor rRNA studies. Analysis of whole‐cell RNA pulse‐labeled with [Me‐3H]methionine, or nucleolar RNA pulse‐labeled with [3H]uridine in the presence of tubercidin (inhibitor of rRNA processing), as well as electron microscopy of high‐molecular‐weight RNA fractions confirm that the 40‐S precursor rRNA (2.8 × 106) is the primary transcript of the ribosomal genes in Xenopus laevis.
Recycling of Glucose by Rat Hepatocytes
Authors: KATZ Joseph , WALS P. A. , GOLDEN Sybil , ROGNSTAD Robert
Published: March 2005

The metabolism of glucose labeled uniformly with 14C, and in positions 2, 3 and 5 with tritium by hepatocytes from fed and fasted rats were studied. Cells were incubated with glucose as sole substrate, or with glucose and a variety of glucose precursors, and uptake or production of glucose, and the utilization of the isotopes was determined.

There was no uptake of glucose at concentration of up to 15 mM, and net glucose synthesis in the presence of precursors. 14C was however recovered in CO2, lactate and amino acids, and tritium in water. Considerable incorporation into glycogen from 14C and 3H‐labeled glucose occurred at high (above 20 mM) glucose concentrations.

The yield in water always exceeded that in 14C‐labeled products. The yield in 3HOH from [2‐3H]glucose exceeded that from [5‐3H]glucose, and the latter was greater than from [3‐3H]glucose.

Utilization of labeled glucose does not follow Michaelis‐Menten kinetics. The fractional rate of uptake of 14C and tritium‐labeled glucose increases with glucose concentration with a maximum at about 15 mM and then declines.

The effect of numerous gluconeogenic substrates on the isotope utilization and the 3H/14C ratio in glycogen was studied. The uptake of 14C was always depressed. Addition of lactate and dihydroxyacetone has little effect on the detritiation of [2‐3H]glucose, but it is depressed by other substrates. The detritiation of [3‐3H]‐ and [5‐3H]glucose is depressed in aluconeogenesis, that from [3‐3H]glucose usually more than from [5‐3H]glucose. In the presence of lactate detritiation of [3‐3H]glucose is about half that from [5‐3H]glucose.

Equations to calculate the phosphorylation of glucose and fructose 6‐phosphate in the presence of futile cycling between glucose and glucose 6‐phosphate and fructose 6‐phosphate and fructose 1,6‐bisphosphate were derived.

The estimate of glucose phosphorylation requires determination of the specific activity of glucose 6‐phosphate from [2‐3H]glucose. It appears that. futile cycling between glucose and glucose 6‐phosphate is extensive in cells with a high glycogen content, but is low in cells from starved rats and nearly absent in those from diabetic animals.

The estimation of the phosphorylation of fructose 6‐phosphate in the presence of cycling requires knowledge of the specific activities of fructose 6‐phosphate and fructose 1,6‐bisphosphate from [3‐3H]glucose. At present there are no adequate data to calculate phosphorylation and recycling of fructose 6‐phosphate, but under some conditions the rate may be quite high.
Affinity Labelling to ‐ SH Groups in Adenosine ‐ Triphosphate ‐ Phosphoribosyl Transferase with the Dinitrophenyl Group from S‐Dinitrophenyl‐6‐mercaptopurine‐riboside 5′‐Phosphate
Authors: DALL‐LARSEN Torbjørn , FASOLD Hugo , KLUNGSØYR Leiv , KRYVI Håkon , MEYER Christa , ORTANDERL Franz
Published: March 2005

Adenosine–triphosphate–phosphoribosyl transferase from Escherichia coli reacts with S‐dinitrophenyl‐6‐mercaptopurine‐riboside 5′‐phosphate. In this reaction the dinitrophenyl group becomes attached to the enzyme, while the nucleotide is split off. Most aliphatic high and low‐molecular‐weight –SH compounds react with the thioether in the opposite way, i.e. bind the nucleotide and split off dinitrothiophenol. It appears that the dinitrophenyl moiety of the thioether interacts with the enzyme in a specific way, and that this interaction activates the bond between the dinitrophenyl group and the sulfur atom. In support of this it was found that dinitrophenol inhibits the transferase reaction with half maximal effect at 0.4 mM. The inhibition is competitive with ATP. Dinitrophenol also competes with ATP in binding studies.
Tubular Polymers Derived from Helix pomatiaβ‐Hemocyanin
Authors: VAN BREEMEN Jan F. L. , WICHERTJES Trijntje , MULLER Marijke F. J. , VAN DRIEL Roel , VAN BRUGGEN Ernst F. J.
Published: March 2005

Upon trypsinolysis Helix pomatia ß‐hemocyanin forms long tubular structures, which appear to be linear polymers of hemocyanin molecules from which the collar structure has been removed. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that only few peptide bonds are hydrolyzed by trypsin. The structure of the polymers has been investigated by electron microscopy, combined with optical diffraction. Preliminary X‐ray diffraction data are presented. Functional properties of the polymers are similar to those of the native protein. Both show a calciumion‐dependent co‐operativity of oxygen binding and a Bohr effect. The results suggest that the collar of a hemocyanin molecule has no special function in the process of (co‐operative) oxygen binding, different from that of the wall of the molecule.
Antigenic Structure of the Cyanogen Bromide Peptide F‐CB3 from Fibrinogen ‐Chain
Authors: LOTTER Helga , TIMPL Rupert
Published: March 2005

The antigenic properties of the cyanogen bromide peptide F‐CB3 from bovine fibrinogen a‐chain were studied in radioimmune assays with rabbit antibodies to fibrinogen or to peptide F‐CB3. Both fibrinogen and peptide F‐CB3 were indistinguishable in inhibition and dissociation tests. Modification of the single disulfide bridge in peptide F‐CB3 either by reduction or by cleavage with cyanide was not accompanied by loss of serologic activity. Inhibition studies with three individual fragments obtained after cyanide cleavage (molecular weight range 7000 to 23000) indicated the presence of at least three distinct antigenic determinants in peptide F‐CB3. After trypsin digestion of peptide F‐CB 3 still 75 % of its maximal inhibiting capacity was retained. Lack of change in antigenic activity of peptide F‐CB3 after release from the fibrinogen molecule by cyanogen bromide and upon further fragmentation is presumably due to the presence of several sequential antigenic determinants but the presence of conformational determinants could not be entirely excluded. Since no cross‐reaction was observed between bovine and human peptides F‐CB3 one may expect considerable variation in their amino acid sequence.
Heterogeneity of Lipopolysaccharides. Analysis of Polysaccharide Chain Lengths by Sodium Dodecylsulfate‐Polyacrylamide Gel Electrophoresis
Authors: JANN Barbara , RESKE Konrad , JANN Klaus
Published: March 2005

Lipopolysaccharide preparations from R (rough) Escherichia coliO8‐, SR (semirough) Salmonella typhimurium and S (smooth) strains E. coli O8 and Citrobacter 396 were disintegrated with sodium dodecylsulfate and subjected to polyacrylamide gel electrophoresis in the presence of 1 % sodium dodecylsulfate. The results obtained were compared with those obtained from the same lipopolysaccharide preparations by degradation analysis. In dodecylsulfate gel electrophoresis the lipopolysaccharide preparation from the E. coli R mutant and the S. typhimurium SR mutant showed one band each (R‐ and SR‐ band, respectively) with different electrophoretic mobilities. The lipopolysaccharide preparation from the E. coli O8‐strain exhibited two bands, one of which had the same electrophoretic mobility as the R‐band and the other was identified as S‐band. The lipopolysaccharide preparation from the Citrobacter 396 S‐strain exhibited four bands: one R‐band, one SR‐band and two S‐bands. The results showed that wild‐type S strains contain more than one type of lipopolysaccharide. They differ in the length of their O‐specific polysaccharide chains. The lipid A content of the different lipopolysaccharide was expressed in their electrophoretic mobilities.
Recognition of Initiation Codons in Modified f2 RNA by Escherichia coli Ribosomes
Authors: SZKOPIŃSKA Anna , ZAGÓRSKI Wlodzimierz , ZAGÓRSKA Ludwika , SZAFRAŃSKI Przemyslaw
Published: March 2005

f2 phage RNA treated with O‐methylhydroxylamine under denaturing conditions loses its ordered structure with consequent exposure of the normally hidden initiation codons. In the presence of Escherichia coli ribosomes and crude initiation factors modified f2 RNA binds about 50 times more f‐[3H]Met‐tRNA than native f2 RNA. The interaction of native f2 [14C]RNA with ribosomes requires initiation factors. The binding of O‐methylhydroxylamine‐modified f2 [14C]RNA to E. coli 70‐S or 30‐S ribosomes does not depend on the presence of initiation factors. A significant number of ribosome‐ deficient in initiation factors interact with a molecule of modified f2 [14C]RNA. Treatment of the resultant polysomal complex with pancreatic RNase yields ribosomes with f2 RNA fragments protected against RNase. Almost all AUG/GUG codons in the f2 RNA are located on the RNaseinsensitive ribosome‐bound fragments, constituting only 25% of the entire molecule. Addition of crude initiation factors to such ribosomes with fragments of modified f2 RNA promotes binding of f‐[3H]Met‐tRNA. The resultant complex is fully reactive with puromycin. No binding of Ac‐PhetRNA takes place under similar conditions.
Translation of Honeybee Promelittin Messenger RNA
Authors: SUCHANEK Gerda , KINDÅS‐MÜGGE Ingela , KREIL Günther , SCHREIER Max H.
Published: March 2005

Venom glands of honeybees synthesize the peptide melittin via the precursor promelittin. Total RNA preparations from venom glands served as template in a cell‐free system prepared from mammalian cells. The heterologous system translated the insect mRNA with approximately the same efficiency as hemoglobin mRNA. A polypeptide was synthesized which, as shown by acrylamide gel electrophoresis in the presence of detergent, has a higher molecular weight than promelittin. Analysis of peptic fragments as well as Edman degradation have demonstrated that sequences characteristic of venom gland promelittin are present in this product formed in vitro. Furthermore, a bacterial protease which specifically splits after acidic residues liberates from the cell‐free product a fragment which closely resembles melittin. Evidence is presented that most of the extra amino acids are located at the amino terminus of the product formed in vitro.

The larger polypeptide detected in vitro may represent a precursor of promelittin.
Phenylalanine Hydroxylase of Macaca irus
Authors: COTTON Richard G. H. , GRATTAN Phillip J.
Published: March 2005

A simple method is described for the purification of phenylalanine hydroxylase using affinity chromatography.

Two forms of enzyme are separated by this method. One of these is only partially purified and the other is highly purified. Attempts to interconvert these forms were unsuccessful.
Kinetics and Effect of Salts and Polyamines on T4 Polynucleotide Ligase
Authors: RAAE Arnt J. , KLEPPE Ruth K. , KLEPPE Kjell
Published: March 2005

The kinetics of T4 polynucleotide ligase has been investigated at pH 8, 20 °C and using the double‐stranded DNA substrate (dA)n [(dT)10]n/10. Double‐reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping‐pong mechanism. The overall mechanism was found to be non‐processive. The true Km for the DNA substrate was 0.6 μM and that of ATP 100 μM.

Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32P‐labelled (dA)n [(dT)40]n/40 as substrate. No breakdown of this DNA could be detected.

The joining reaction was inhibited by high concentrations, i.e. above approximately 70 mM, of salts such as KC1, NaCl, NH4Cl and CsC1. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.
Glutathione Reductase from Human Erythrocytes
Authors: WORTHINGTON David J. , ROSEMEYER Michael A.
Published: March 2005

Glutathione reductase from human erythrocytes exists predominantly as an entity of 100000 molecular weight under various conditions of pH and ionic strength. The s20,w of 5.5 S and D20,w of 50 μm2/s correlate with the molecular weight determined by sedimentation equilibrium. The homogeneity of this species is primarily dependent on the presence of thiols and secondarily on high concentrations of salt.

The amino‐acid composition of the enzyme shows similarities both with glutathione reductases from other sources and with lipoamide dehydrogenase.

From the flavin content and dodecylsulphate‐polyacrylamide electrophoresis it is inferred that the native enzyme is a dimer composed of similar subunits of 50000 molecular weight.

In the absence of thiols, glutathione reductase shows a tendency to form tetramers and larger aggregates.

Although these larger species are also catalytically active, under cellular conditions the presence of its product, reduced glutathione, should maintain the enzyme as the dimeric entity.