bluuebunny/crossref_metadata_2025 huggingface dataset:
A Rare Genetically Determined Variant of Pseudocholinesterase in two German Families with High Plasma Enzyme Activity
Authors: DELBRÜCK Axel , HENKEL Eberhard
Published: March 2005

Activity of pseudocholinesterase (acylcholine‐acyl‐hydrolase) elevated up to four times has been detected in sera of members of two German families. The catalytic concentrations of the pseudocholinesterase of the afflicted members of both families (male and female) varied between 4800 U/l and 10200 U/l (acetylthiocholine iodide substrate). The pseudocholinesterase of the propositi exhibits isoenzyme separation patterns in polyacrylamide electrophoresis as well as in electrofocussing which are different from those of pseudocholinesterase from normal persons. No differences could be seen as regards the Km of substrates or the inhibition by dibucaine, fluoride or succinyldiocholine.
Interaction of Butene with Human Hemoglobin A
Authors: POYART Claude , BURSAUX Elisabeth , GUESNON Patrick , BOHN Brigitte
Published: March 2005

The binding of various alkanes by proteins was recognized years ago. We have studied the effect of butene (C4H8), a short‐chain aliphatic hydrocarbon, on the functional properties of human adult hemoglobin. Under 1 atm pressure (100 kPa) butene decreased the affinity of hemoglobin (Hb) for oxygen (p50) by 45% without altering the cooperativity of ligand binding. This effect was independent of pH (from 7.0 to 8.0) and of ionic strength. The changes in the affinity of hemoglobin for oxygen were dependent upon the partial pressure of butene and evoked a saturating mechanism of the binding site(s). Mathematical simulation of the curve relating p50 to the concentration of dissolved butene allowed us to calculate the apparent association constants for one single binding site KHb= 10.4 mmol‐1 and KHbO2= 1.53 mmol‐1 to Hb and HbO2 respectively. The larger binding of butene by Hb was confirmed by a 25% decrease in K1, the first association constant of oxygen to the tetrameric hemoglobin. It is concluded that butene is an allosteric effector of human Hb which acts most likely through hydrophobic interactions. It is postulated that the oxygen‐linked binding site may be located at the α1β2 interface.
Binding of Specific ATP Citrate Lyase and Fatty Acid Synthetase Antibodies to Heavy Populations of Rat Liver Polysomes
Authors: FINKELSTEIN Mary B. , AURINGER Michael P. , HALPER Laura A. , LINN Tracy C. , SINGH Manoranjan , SRERE Paul A.
Published: March 2005

The polysome fractions involved in the synthesis of the rat‐liver inducible lipogenic enzymes, ATP citrate lyase and fatty acid synthetase, were identified by their binding of radioiodinated specific antibodies to enzyme. Both of these populations of specific polysomes were shown to be markedly heavier than the average rat liver polysomes and significantly heavier than specific polysomes involved in albumin synthesis. The quantity of antibody bound to the lipogenic enzyme‐related polysomes was markedly affected by the dietary status of the animal. A dietary regimen which induced lipogenesis resulted in a tenfold increase in the hepatic activities of these enzymes found in normally fed animals. The radioactivity bound to hepatic polysomes of induced rats was likewise greater than tenfold higher, presumably reflecting an increase in the number of polysomes active in enzyme synthesis. The fasting state resulted in lower hepatic enzyme activity than normal and correspondingly less binding of ATP citrate lyase and fatty acid synthetase antibodies to the heavy polysomes of the sucrose gradient.
Characterization of Pre‐messenger‐RNA‐Containing Nuclear Ribonucleoprotein Particles from Avian Erythroblasts
Authors: MAUNDRELL Kinsey , SCHERRER Klaus
Published: March 2005

Ribonucleoprotein particles have been isolated from duck erythroblast nuclei using a procedure designed to produce maximal cytoplasmic dispersion with minimal release of endogenous hydrolytic enzymes. The RNA extracted from the purified nuclear ribonucleoprotein fraction is shown to contain globin messenger RNA sequences at a concentration comparable to that present in total nuclear RNA. The polypeptide composition of this fraction revealed by electrophoresis in two dimensions is complex, consisting of at least 65 acidic species and 21 basic species. Several lines of evidence suggest that these are authentic components of nuclear ribonucleoprotein. The so‐called “core” proteins of nuclear ribonucleoprotein which were previously shown to migrate as a single band on low‐pH urea gels, and as six bands on sodium dodecyl sulphate gels are here shown to be considerably more complex being resolved by two‐dimensional electrophoresis into a group of 15 basic and 6 more and less neutral polypeptides. Isoelectric focusing of nuclear ribonucleoprotein under non‐denaturing conditions suggests that these latter species are not uniformly distributed along the pre‐messenger RNA molecule.
A Fluorescamine‐Based Sensitive Method for the Assay of Proteinases, Capable of Detecting the Initial Cleavage Steps of a Protein
Authors: GARESSE Rafael , CASTELL José V. , VALLEJO Carmen G. , MARCO Roberto
Published: March 2005

The properties of the reaction of fluorescamine with proteins are the basis for the development of a sensitive, general and simple method for the assay of proteolytic activities. More importantly, the assay measures the initial step(s) of proteolytic attack, making it specially suitable for the examination of the controlling factors that regulate proteolytic degradation and/or the detection of “specific” proteinases. The method allows the simple determination of the general parameters of enzyme action, V and Km, using proteins, i.e. the physiological substrates of the proteinases. The more appropriate proteins to be used as substrates are the N‐amino‐terminally blocked ones. Many proteins fulfill this requirement. If the particular protein whose degradation has to be studied lacks this modification, three different approaches can be used to study its degradation: (a)the accumulation of N‐amino termini in excess over that of the intact substrate; (b)a gel filtration/continuous method and (c) the chemical blockage of its amino groups. The particular advantages of each of these approaches are discussed.
Identification of the C‐1‐Phosphate‐Binding Arginine Residue of Rabbit‐Muscle Aldolase
Authors: PATTHY László , VÁRADI András , THÉSZ János , KOVÁCS Katalin
Published: March 2005

The arginine‐specific reagent 1,2‐cyclohexanedione reacts selectively with the arginine residue of the C‐1‐phosphate‐binding site of aldolase and inactivates the enzyme. The labeled peptide isolated from tryptic digests of inactivated aldolase was found to correspond to the sequence Leu‐43 to Arg‐56, the residue modified by cyclohexanedione being Arg‐55. This peptide was absent from digests of aldolase treated in the same way but protected from inactivation by the presence of substrate, thus correlating modification of Arg‐55 with loss of activity.

Selective isolation of the peptide containing the modified arginine residue was effected by chemisorption chromatography on boric acid gel, a procedure exploiting the specific interaction of matrix‐bound boric acid groups with vicinal m‐hxdroxyl groups of cyclohexanedione‐modified arginine side chains.
Fluorescently Labelled Histones as Probes of Nucleosome Structure
Authors: LEWIS Peter N.
Published: March 2005

A fluorescent derivative of calf thymus histone H4 has been prepared by the reaction of methionine‐84 with N‐(iodoacetylaminoethyl)8‐naphthylamine‐1‐sulfonic acid at pH 2.4 in 8 M urea. The preparation and characterization of this labelled histone is described. Fluorescence emission measurements indicate that the label onH4 undergoes a 3–5‐fold increase in emission intensity when H4 self‐interacts or binds to DNA alone or is incorporated in a synthetic nucleosome. The changes observed are consistent with the formation of varied apolar environments around methionine‐84, due most likely to histone‐histone rather than histone‐DNA interactions. Preliminary experiments indicate that the precise emission intensity of labelled H4 in the nucleosome is quite sensitive to conditions of ionic strength and histone integrity.
The Two Major Isozymes of Yeast Alcohol Dehydrogenase
Authors: WILLS Christopher , JÖRNVALL Hans
Published: March 2005

The structures of the two cytoplasmic isozymes of alcohol dehydrogenase in a haploid strain of the yeast Saccharomyces cerevisiae have been compared and related to a commercial preparation of the protein that was previously analyzed. The constitutive isozyme I (purified from a petite cell incapable of mitochondrial respiration) is found to correspond closely to the commercially available but taxonomically uncharacterized enzyme. After analysis of 86 % of all positions, two amino acid differences were found among the 298 residues compared. The enzymes from these sources are thus about 99 % identical.

The inducible isozyme II (purified from aerobically grown grande cells) differs substantially from isozyme I. After analysis of 82% of all positions, there are 15 amino acid exchanges in relation to isozyme I, and 17 in relation to the commercial preparation among the 284 residues compared. Some of the differences established between isozymes I and II explain heterogeneities previously observed in yeast alcohol dehydrogenase.

The amino acid sequences can be matched without insertions or deletions, and 15 of the 17 differences are interpretable as single base changes. The close homology of the two isozymes shows that they were produced from a gene duplication which is unexpectedly recent in view of the functional differences of the isozymes.

Differences in patterns of subunit cooperativity and in Km values have been found between the isozymes. Reflecting this, four of the differences between forms I and II appear to correspond to positions close to the interface between subunits in the tertiary structure of the related liver alcohol dehydrogenase, while two apparently correspond to residues near the inter‐domain cleft of that subunit. No residues with known catalytic functions are, however, exchanged.

Normally, the amino terminus of the yeast enzyme is blocked by an acyl group, but a significant amount of the isozyme II preparation was found to have a free amino terminus. The presence of unblocked molecules is very rare among acetylated proteins, but may lead to further insight into possible roles of α‐amino‐acetylation.
Synthesis and Characterization of a DNA Complementary to Pre‐uteroglobin mRNA
Authors: ARNEMANN Jutta , HEINS Bärbel , BEATO Miguel
Published: March 2005

Uteroglobin, a progesterone‐induced uterine protein of the rabbit, is synthesized in cell‐free systems as a precursor containing 21 additional amino‐acids at its N‐terminal end. The mRNA for pre‐uteroglobin has been purified from the membrane‐bound polysomes of induced endometrium and used as template for the synthesis of a full copy complementary DNA. Final purification of the cDNA was based on hybridization to the template mRNA up to a low value of r0t(0.01 M · s) and digestion of the non‐hybridized cDNA by S1 nuclease.

A comparison of the hybridization kinetics of the pre‐uteroglobin cDNA and rabbit globin cDNA to their respective templates indicates a nucleotide sequence complexity of 650 for pre‐uteroglobin mRNA, in agreement with the values obtained by sucrose gradient centrifugation and polyacrylamide gel electrophoresis in formamide. The melting temperature of the hybrids of pre‐uteroglobin cDNA to its template reflects the absence of mismatched sequences. This cDNA has been used to quantify pre‐uteroglobin mRNA sequences in the endometrial RNA from control animals and from animals treated sequentially with estradiol and progesterone. In agreement with the induction of uteroglobin‐synthesizing activity, there is a dramatic increase in the uterine content of pre‐uteroglobin mRNA after hormonal treatment. Part of this effect can be accounted for by hormonally induced cell proliferation. When expressed on a DNA basis there is a 50–100‐fold increase in the cellular content of pre‐uteroglobin mRNA following hormonal treatment.
Netropsin · dG‐dG‐dA‐dA‐dT‐dT‐dC‐dC Complex
Authors: PATEL Dinshaw J.
Published: March 2005

The structure of the netropsin · dG‐dG‐dA‐dA‐dT‐dT‐dC‐dC complex (one antibiotic molecule/ self‐complementary octanucleotide duplex) and its dynamics as a function of temperature have been monitored by the nuclear magnetic resonances of the Watson‐Crick protons, the nonexchangeable base and sugar protons and the backbone phosphates. The antibiotic forms a complex with the nucleic acid duplex at the dA · dT‐containing tetranucleotide segment dA‐dA‐dT‐dT, with slow migration amongst potential binding sites at low temperature. The downfield shifts in the exchangeable protons of netropsin on complex formation demonstrate the contributions of hydrogen‐bonding interactions between the antibiotic and the nucleic acid to the stability of the complex. Complex formation results in changes in the glycosidic torsion angles of both thymidine residues and one deoxyadenosine residue as monitored by chemical shift changes in the thymine C‐6 and adenine C‐8 protons. The close proximity of the pyrrole rings of the antibiotic and the base‐pair edges in the minor groove is manifested in the downfield shifts (0.3–0.5 ppm) of the pyrrole C‐3 protons of netropsin and one adenine C‐2 proton and one thymine N‐3 base‐pair proton on complex formation. The internucleotide phosphates of the octanucleotide undergo 31P chemical shift changes on addition of netropsin and these may reflect, in part, contributions from electrostatic interactions between the charged ends of the antibiotic and the backbone phosphates of the nucleic acid.
Determination by Radioimmunoassay of the Sum of Oxidized and Reduced Forms of NAD and NADP in Picomole Quantities from the Same Acid Extract
Authors: BREDEHORST Reinhard , LENGYEL Helgard , HILZ Helmuth
Published: March 2005

The sum of the amounts of NAD + NADH was determined from the same acid tissue extract with the aid of a highly specific radioimmunoassay for 5′‐AMP. NAD was converted to 5′‐AMP via ADP‐ribose by alkaline treatment while NADH was converted first to ADP‐ribose by incubation of the acid extract at 25°C followed by alkaline conversion to 5′‐AMP. Removal of phosphate groups in NADP and NADPH by treatment of the extracts with alkaline phosphatase extended the procedure to the quantification of NADP(H). When combined with enzymic analyses of the oxidized coenzyme forms, NAD/NADH and NADP/NADPH ratios could also be obtained from the same extracts. The sensitivity of the test allows quantification of pyridine nucleotides in the range of 0.1–10 pmol.
Regulation of Transcription by DNA‐Bound Non‐histone Nuclear Proteins
Authors: CREPIN Michel , DASTUGUE Bernard
Published: March 2005

Purified non‐histone proteins from mouse mammary cells bind specifically to homologous DNA or chromatin. Complexes of non‐histone protein with DNA or chromatin, isolated on agarose columns, were transcribed with both Escherichia coli RNA polymerase and RNA polymerase B from calf thymus. The fact that complexing of DNA with non‐histone proteins increases transcription by E. coli RNA polymerase but not by RNA polymerase B suggests different mechanisms of transcription by these two enzymes. Similar experiments with mouse and Drosophila chromatin indicate that non‐histone proteins specifically stimulate the transcription of mouse chromatin by RNA polymerase B. Non‐histone proteins stimulate the transcription of mouse mammary tumor virus sequences in chromatin by RNA polymerase B but not by E. coli RNA polymerase. We conclude that those non‐histone proteins bound specifically to chromatin are able to activate the transcription of specific genes by eukaryotic RNA polymerase.
Kinetic Mechanism of NADH‐Dependent Glutamate Synthase from Lupin Nodules
Authors: BOLAND Michael J.
Published: March 2005

From initial‐rate studies, a partially random kinetic mechanism has been deduced for NADH‐dependent glutamate synthase from lupin nodules. The mechanism involves compulsory binding of NADH as first substrate, followed by random‐order binding of glutamine and 2‐oxoglutarate. Patterns of inhibition by glutamate substantiate the mechanism. Dithionite was incapable of acting as an alternative reducing substrate although it is known to reduce the flavine groups of the enzyme. The implications of these results are discussed.

Published rate equations for this type of mechanism were found to be unsatisfactory for this enzyme and suitable new equations are produced. These equations should have general application where the obligatory first substrate binds very tightly.
Metabolism of Isolated Kidney Tubules
Authors: GUDER Walter G. , WIRTHENSOHN Gabriele , Pürschel Susanne , Gerl Margit
Published: March 2005

Kidney‐cortex tubule suspensions were prepared by collagenase treatment of kidney cortex from fed and starved rats. This preparation, consisting mainly of proximal convoluted tubules was incubated with three major renal substrates, L‐lactate, glutamine and oleate to study the dose dependence of substrate uptake rates from medium substrate combinations.

All three substances, when added at near physiological concentrations, modified the uptake rate and fate of the other substrates. In accordance with previous observations, oleate inhibited lactate uptake, and lactate decreased glutamine metabolism. Glutamine on the other hand led to a marked increase in lactate uptake. Both, glutamine and lactate increased oleate metabolism. Glucose was the main product of lactate and glutamine metabolism, lactate being preferentially taken up for this process.

Oleate led to a net synthesis of triglycerides in the tubules, which was stimulated by the addition of lactate and glutamine. More than 75 % of the oleate taken up was recovered as triglycerides. In the absence of fatty acids, triglyceride content of tubules decreased.

The results indicate that oleate is taken up in preference to lactate and glutamine when all three substrates are offered to the tubule. Glucose and triglycerides are the main metabolic products of tubular substrate metabolism. Whereas glucose is released into the medium, triglycerides are stored in the tubule cell.
Characterization of Bovine Anterior‐Lens‐Capsule Basement‐Membrane Collagen
Authors: SCHWARTZ David , VEIS Arthur
Published: March 2005

Collagen extracted from bovine anterior lens capsule basement membrane under minimally degradative conditions can be precipitated in the form of segment‐long‐spacing crystallites. Positive and negative stain preparations of these precipitates, using uranyl acetate, show the crystallites to have an asymmetric band pattern, indicating unidirectional orientation of all molecules in each crystallite. Non‐banded globular masses appear at both ends of most of the crystallites, giving the segment‐long‐spacing aggregates the form of dumbells, with a total length of 300 nm. Repeated pepsin digestion removes first one then both globular regions leaving the helical region intact but with a length still equal to 300 nm. It thus appears that the pepsin‐susceptible protein is folded back over and covers the ends of the helical regions. Preparations with the most intact end regions tend to aggregate in fibrous segment‐long‐spacing form, that is, in end‐interacted strings or nets. The very prominent molecular end‐region interaction suggests a similar filamentous but non‐periodic organization within native basement membrane.
Phosphate Binding to Liver Alcohol Dehydrogenase Studied by the Rate of Alkylation with Affinity Labels
Authors: DAHL Knut H. , MCKINLEY‐MCKEE John S.
Published: March 2005

In this work we report that phosphate anions interact with the anion binding site of alcohol dehydrogenase from horse liver. In protection experiments against the two affinity labels, iodoacetic acid and bromo‐imidazolylpropionic acid, the dissociation constant for the enzyme‐phosphate complex at pH 7.0 is, based on total phosphate, found to be 20 ± 5 mM. The 1,4‐piperazinediethanesulfonate anion has a lower affinity for the anion binding site, the dissociation constant at pH 7.0 being 130 ± 20 mM. The anion‐independent dissociation constants for the reversible enzyme‐affinity label complexes are at pH 7.0, 1.35 ± 0.2 mM for iodoacetic acid and 0.39 ± 0.05 mM for bromo‐imidazolylpropionic acid. These findings have important implications with respect to past and future work on this well known enzyme.
Studies on the Intracellular Distribution of Sindbis Messenger RNA in Infected Chick Embryo Fibroblasts
Authors: BORGESE Nica , MELDOLESI Jacopo , BONATTI Stefano , CANCEDDA Ranieri
Published: March 2005

The submicrosomal distribution of membrane‐associated viral 26‐S RNA in chick embryo fibroblasts infected with Sindbis virus was studied. Infected chick embryo fibroblasts were homogenized in the presence of low amounts of EDTA and fractionated by differential centrifugation. Analysis of postmitochondrial supernatants by isopycnic flotation on continuous sucrose gradients showed that membrane‐associated 26‐S RNA and ribosomes were not distributed in parallel, with an enrichment in 26‐S RNA in the light microsomal subfractions. This distribution could not be explained by adsorption artifacts. Analysis of the distribution of microsomal constituents on sucrose gradients after treatment with digitonin ruled out the possibility that the 26‐S RNA might be associated with Golgi or plasma membranes. The attachment of viral RNAs to endoplasmic reticulum membranes is discussed in relation to its possible role in viral morphogenesis.
Shut‐Off of Host Protein Synthesis in Vaccinia‐Virus‐Infected Cells Exposed to Cordycepin
Authors: PERSON Anne , BEAUD Georges
Published: March 2005

Cytoplasmic lysates from Ehrlich ascites tumor cells were prepared for very efficient endogenous protein synthesis in vitro. It was shown that initiation of polypeptide chain occurred in vitro since 70–85% of protein synthesis in vitro was inhibited by aurintricarboxylic acid or edeine, it was also calculated that about three protein chains were synthesized per active ribosome from the amount of [35S]methionine incorporated into newly synthesized proteins. The time of centrifugation of the cell lysate used for preparing the cytoplasmic extract was important in obtaining efficient protein synthesis in vitro. When cytoplasmic extracts were prepared from vaccinia‐virus‐infected cells exposed to cordycepin (3′‐deoxyadenosine), an inhibition of 50–70% of the endogenous protein synthesis in vitro was observed, similar to the inhibition seen in vivo. Although the incorporation of formylated methionine from f[35S]Met‐tRNAMetf was not inhibited in the infected cell extracts, the incorporation of [35S]methionine into the N‐terminal position was significantly reduced; since [35S]methionine incorporation (into protein) sensitive to aurintricarboxylic acid and edeine was also reduced in the infected cell extracts, it was concluded that the initiation of polypeptide chains in vitro was inhibited in the vaccinia‐virus‐infected cell extracts. The addition of ribosomal salt wash and spermine to the extracts, although inducing a two–fourfold stimulation of the endogenous protein synthesis in both extracts, did not release the inhibition observed in the infected cell extracts. When the lysates were pretreated with micrococcal nuclease, an inhibition of mRNA‐dependent protein synthesis of approximately 50% was observed in the extracts prepared from vaccinia‐virus‐infected cells exposed to cordycepin. This inhibition occurred either with globin mRNA or with early vaccinia virus mRNA, confirming that the inhibitory factor associated with vaccinia virus did not discriminate between cellular and viral mRNA.
Two Distinct Poly(A) Polymerases Isolated from the Cytoplasm of Ehrlich Ascites Tumour Cells
Authors: AVRAMOVA Zoya V. , MILCHEV Georgui I. , HADJIOLOV Asen A.
Published: March 2005

The poly(A) polymerases from the cytosol and ribosomal fractions of Ehrlich ascites tumour cells are isolated and partially purified by DEAE‐cellulose and phosphocellulose column chromatography. Two distinct enzymes are identified: (a) a cytosol Mn2+‐dependent poly(A) polymerase (ATP: RNA adenylyltransferase) and (b) a ribosome‐associated enzyme defined tentatively as ATP(UTP) :RNA nucleotidyltransferase.

The cytosol poly(A) polymerase is strictly Mn2+‐dependent (optimum at 1 mM Mn2+) and uses only ATP as substrate, Poly(A) is a better primer than ribosomal RNA. The purified enzyme is free of poly(A) hydrolase activity, but degradation of [3H]poly(A) takes place in the presence of inorganic pyrophosphate. Most likely this enzyme is of nuclear origin.

The ribosomal enzyme is associated with the ribosomes but it is found also in free state in the cytosol. The purified enzyme uses both ATP and UTP as substrates. The substrate specificity varies depending on ionic conditions: the optimal enzyme activity with ATP as substrate is at 1 mM Mn2+, while that with UTP as substrate is at 10–20 mM Mg2+. The enzyme uses both ribosomal RNA and poly(A) [but not poly(U)] as primers. The purified enzyme is free of poly(A) hydrolase activity.
Primer Specificity of Ribosome‐Associated Poly(A) Polymerase from Ehrlich Ascites Tumour Cells
Authors: MILCHEV Georgui I. , AVRAMOVA Zoya V. , HADJIOLOV Asen A.
Published: March 2005

The reaction product of the ribosomal poly(A) polymerase [ATP(UTP):RNA ucleotidyltrasferase] is analyzed. Two systems are used in vitro: (a) isolated polyribosomes with endogenous enzyme and RNA primer and (b) purified enzyme with total polyribosomal RNA as primer. In the polyribosome system about 50% of the [3H]AMP label is in poly(A)‐containing mRNA. This RNA displays a heterogeneous size ditribution in the range of 8–30 S with a maximum at about 14 S. Upon denaturation the maximum is shifted towards the 10–S zone. The poly(A) polymerase catalyzes the addition of 12–18 adenylate residues to pre‐existing mRNA poly(A) sequences of 40–160 residues. The [3H]AMP incorporated into poly(A)‐lacking RNA is mainly in a fraction with an electrophoretic mobility corresponding to 4‐S RNA. In the purified enzyme system, specificity towards poly(A)‐containing mRNA is lost to a considerable extent. Only 10% of the [3H]AMP label is retained by oligo(dT)‐cellulose. The bulk of the product is in 18‐S rRNA and heterogeneous small molecular weight RNA.

We conclude that the ribosome‐associated poly(A) polymerase is most likely the enzyme responsible for the cytoplasmic polyadenylation of poly(A)‐containing mRNA in vivo.
The Energy‐Dependent Degradation of Guanosine 5′‐Diphosphate 3′‐Diphosphate in Escherichia coli
Authors: TÉTU Chantal , DASSA Elie , BOQUET Paul‐L.
Published: March 2005

Different inhibitors of the energy metabolism have been assayed in Escherichia coli K12 for their ability to increase the level of guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) as a consequence of a restriction of its degradation. Inhibitors of the respiration and uncouplers of oxidative phosphorylations had effects similar to carbon‐source‐induced downshifts while the ATPase inhibitor dicyclohexylcarbodiimide was less efficient. The effects of dicyclohexylcarbodiimide and of the uncoupler carbonylcyanide p‐fluoro methoxyphenylhydrazone (FCCP) on ppGpp degradation were compared in a drug‐permeable envelope mutant. At concentrations of inhibitors sufficient to deplete the pool of ATP by 50%, only FCCP was able to block ppGpp degradation. Moreover, FCCP also inhibited ppGpp degradation in a ATPase‐deficient strain growing on glucose as carbon source while, as expected, it did not change the level of ATP. It is concluded, according to Mitchell's chemiosmotic hypothesis, that, in vivo, the integrity of the transmembrane proton gradient rather than the ATP pool size is a prerequisite for the normal processing of the energy‐dependent degradation of ppGpp.
Fluorimetric Study of the Complex between Yeast Phenylalanyl‐tRNA Synthetase and tRNAPhe
Authors: LEFEVRE Jean‐François , EHRLICH Ricardo , REMY Pierre
Published: March 2005

The variations of several spectroscopic properties of yeast tRNAPhe and phenylalanyl‐tRNA synthetase upon complex formation, were used to study the stoichiometry of the complex in different experimental conditions.

In all cases, for the tRNAPhe‐enzyme complex, in the absence of other ligands, the saturations of the different conformational changes monitored for both macromolecules, are achieved at a 2:1 tRNA/enzyme stoichiometry. Phenylalanine does not modify this saturation. In contrast, the presence of 1 mM ATP induces an asymmetric behaviour of the synthetase: two tRNAs are still bound per enzyme molecule but the conformational change of the latter is completed upon binding of a single tRNA molecule.
Chain Inequivalence in Bovine Methemoglobin
Authors: ILAN Yael A. , ILAN Yigal , CHEVION Mordechai , CZAPSKI Gidon
Published: March 2005

Using pulse radiolysis, a single heme in the tetramer of bovine methemoglobin was reduced within a few microseconds to the ferro state, producing a valence intermediate. The kinetics of oxygen binding to the valence intermediate as well as the re‐oxidation of the ferro‐heme to the ferric state were studied as a function of pH.

The kinetics of the oxygenation revealed the existence of two species, characterized by high and low affinities for oxygen that are associated with two quaternary structures (R and T, respectively). A sigmoidal curve representing a transition between the two states as a function of pH was derived. Above pH 7.7 only the R state could be observed, while below pH 6.5 the T state was dominant.

The reaction between the valence intermediate and ferricyanide at pH 7.75 (R state) consisted of two (about) equal contributions (k1= 23 × 104M−1 s−1; k2= 2.1 × 104 M−1 s−1) attributed to the β and α subunits within the tetramer, respectively. At pH 6.3 (T state) a similar phenomenon was observed (k1= 69 × 104 M−1 s−1; k2= 3.7 × 104 M−1 s−1), indicating chain inequivalences both in the T and the R states of methemoglobin.

In the presence of inositol hexakisphosphate the T → R transition, as monitored by oxygenation of the valence intermediate, was shifted up to a higher pH by about 0.35. Yet similar rate constants exhibiting similar chain inequivalences have been measured.
Arginyl‐Transfer Ribonucleic Acid Synthetase of Bacillus stearothermophilus. Purification and Kinetic Analysis
Authors: GODEAU Jean‐Marie
Published: March 2005

Arginyl‐tRNA synthetase from Bacillus stearothermophilus (NCA 1518) has been purified 880‐fold to apparent homogeneity as demonstrated by electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight is 59000 as confirmed by Sephadex G‐100 and by sucrose gradient ultracentrifugation. The enzyme is monomeric, no subunits were detected. Its cognate tRNA induces an apparent increase in molecular weight suggesting the dimerisation of the enzyme. Nevertheless, it is not obvious that the enzyme dimer forms prior to the aminoacylation reaction catalysed by the enzyme. An ATPase activity was found associated to the synthetase but can be neglected because the ATP consumption is too low for hampering the arginyl‐tRNA synthetase activity.

The order of addition of substrates and release of products has been studied by measurements of initial velocity, product inhibition and dead‐end inhibition. The nature of the kinetic patterns indicates that the aminoacylation reaction conforms to the classical concept of the mechanism which includes the formation of an enzyme‐bound aminoacyl‐adenylate as an intermediate in the first step followed by transfer of the amino acid to tRNA. The first partial reaction, measured by the ATP‐32PPi exchange or AMP synthesis in the presence of ATP and arginine, requires tRNA, which is consistent with the model in which tRNAArg is an activator of the arginyladenylate synthesis.
Studies on Biosynthesis, Assembly and Expression of Human Major Transplantation Antigens
Authors: ALGRANATI Israel D. , MILSTEIN César , ZIEGLER Andreas
Published: March 2005

Biosynthesis and regulation of expression of transplantation antigens as detected by a monoclonal antibody to HLA‐A,B,C antigens (human leucocytic antigen) and a polyclonal antiserum to β2‐micro‐globulin have been investigated using radioactive amino acids and sugars to label human lymphoid cells. We found unbalanced synthesis of HLA heavy chains and β2‐microglobulin, the latter being in excess and secreted to the extracellular medium. In DAUDI cells, which are defective in β2‐microglobulin, no HLA‐A,B,C could be detected intracellularly even in the presence of added β2‐microglobulin. Treatment of BRI‐8 cells with tunicamycin, an antibiotic which inhibits glycosylation of polypeptides, almost had no effect on the levels of β2‐microglobulin, while it markedly decreased that of HLA heavy chains, both on the cell surface and intracellularly. Glycosylation of the HLA heavy chains appeared to be an essential requirement for the normal expression of HLA‐A,B,C antigens. The translation in vitro in a messenger‐dependent reticulocyte system with total polysomes obtained from BRI‐8 cells showed that β2‐microglobulin was synthesized as a precursor. This larger polypeptide was converted into mature β2‐microglobulin when protein synthesis was performed with microsomes instead of polysomes.
Interaction between Major Outer Membrane Protein (O‐8) and Lipopolysaccharide in Escherichia coli K12
Authors: YAMADA Hisami , MIZUSHIMA Shoji
Published: March 2005

An ordered hexagonal lattice structure with a lattice constant of about 7nm was reconstituted from outer membrane protein O‐8 and/or O‐9 and lipopolysaccharide [H. Yamada and S. Mizushima, J. Bacteriol. 135, 1024–1031 (1978)]. Using this reconstitution system, the interaction between O‐8 and lipopolysaccharide was studied and the following results were obtained.

At least one lipopolysaccharide molecule per one O‐8 trimer was required for the hexagonal lattice formation. The lattice constant of the hexagonal lattice formed under the conditions was about 6.5 nm. In the presence of a larger amount of lipopolysaccharide, the lattice constant increased to 7.5 nm, being almost the same as that observed in the dodecylsulfate‐treated cell envelope.

The lattice constant observed with the heptoseless lipopolysaccharide was also 6.5 nm when the lipopolysaccharide/O‐8 ratio was low. Although the increase of the lattice constant was observed upon further addition of the lipopolysaccharide, it was appreciably smaller than that with the wild‐type lipopolysaccharide.

A hexagonal lattice was also formed when lipopolysaccharide was replaced by an equivalent amount of lipid A or even fatty acids. The lattice constant was the same as that with the wild‐type lipopolysaccharide when the lipid/protein ratio was low. However, contrary to the case of lipopolysaccharide, the lattice constant remained almost unchanged even when a larger amount of either lipid A or fatty acid was added to the reconstitution system. A slight increase in the lattice constant was observed only when an extremely large amount of lipid A was added to the reconstitution system.

These results suggest that (1) both the lipid A moiety and the polysaccharide moiety of lipopolysaccharide participate in the interaction with the O‐8 trimer, (2) in the lipid A moiety the fatty acid region primarily participates in the hexagonal arrangement of the O‐8 trimer and the minimum requirement of lipopolysaccharide for the lattice formation is most probably one lipopolysaccharide molecule per O‐8 trimer and (3) the polysaccharide moiety, probably both the 3‐deoxy‐d‐manno‐octulosonate region and the distal end region including heptose, are involved in an interaction of lipopolysaccharide with the O‐8 trimer to give a proper lattice conformation.
Loops in Viroids
Authors: WILD Ursula , RAMM Karla , SÄNGER Heinz Ludwig , RIESNER Detlev
Published: March 2005

The structure of the potato spindle tuber viroid (PSTV) was investigated by binding of specific tRNAs to the molecule. Instead of oligonucleotides tRNAs had to be used for those studies because of their higher binding constants and the easier methods of monitoring the extent of binding. Complex formation was detected by analytical ultracentrifugation and by optical titrations. Buffer conditions were 0.05 M sodium cacodylate, 0.01 M MgCl2, pH 6.8 in the optical titrations, and 0.1 M NaCl was added in the ultracentrifugation experiments.

From sedimentation analysis at 10°C following results were obtained: tRNALys from Escherichia coli, tRNAphe, tRNAAsp, and tRNASer from yeast bind to PSTV with binding constants in the order of 105 M−1. No binding could be detected with tRNAfMet and tRNA1Val from E. coli.

In spectrophotometric titrations with tRNA2Glu and tRNALys from E. coli the decrease in the absorption of mnm5s2U at 300 nm due to the binding was observed. 1 mol tRNA2Glu/mol PSTV binds with a constant of 5.3 × 105 M−1 at 10°C; 3 mol tRNALys/mol PSTV bind with an average constant of 7.0 × 104 M−1 at 20°C.

In fluorimetric titrations with tRNAphe from yeast the quenching of the fluorescence intensity of the wye base due to the binding was monitored. 2 mol tRNAphe/mol PSTV bind with an average constant of 7.5 × 105M−1 at 10°C.

From the experimental results the following conclusions can be drawn. The anticodons of several specific tRNAs bind to complementary sequences in certain loop regions of PSTV. The loop regions determined by this method are in accordance with the nucleotide sequence and the secondary structure model of PSTV as published recently [2,4]. The bases in the internal loops must have a sufficient degree of flexibility to assume a spatial arrangement which is optimal for base‐pairing with other RNAs. The free accessibility of loops distributed over the entire PSTV molecule clearly demonstrates the absence of tertiary structure folding in PSTV.
Further Characterization of the Phosphate Moiety of the Adenovirus Type 2 DNA‐Binding Protein
Authors: LINNÉ Tommy , PHILIPSON Lennart
Published: March 2005

The adenovirus type 2 DNA‐binding protein is phosphorylated. Alkaline phosphatase treatment removes phosphate groups resulting in a decrease in molecular weight from 72000 to 70000. The dephosphorylated protein binds to single‐stranded and double‐stranded DNA as well as the phosphorylated protein does. Controlled chymotrypsin treatment cleaves the DNA‐binding protein into two subspecies of Mr about 45000 and 25000. The 45000‐Mr polypeptide contains most of the methionine residues but no phosphate and binds to DNA. The 25000‐Mr polypeptide contains all the phosphate groups and shows no binding to DNA. Isoelectric focusing gels show heterogeneity of the DNA‐binding protein and 15 subspecies with different charges can be observed after partial dephosphorylation by alkaline phosphatase. After extensive dephosphorylation two or three basic species with a molecular weight around 70000 are observed. Quantitative immunoprecipitation from cells labeled to equilibrium with inorganic 32PO4 gives a molar ratio of phosphate to protein of 4–7 and direct chemical determination of the phosphate residues yields 4 mol Pi/mol protein. These results suggest that there exist subspecies of the protein moiety of the adenovirus DNA‐binding protein.

The DNA‐binding protein isolated from infected cells after a short ‘pulse’ of [35S]methionine has a molecular weight which corresponds to that of the dephosphorylated protein. After a ‘chase’ period the molecular weight increases to 72000, but alkaline phosphatase treatment converts it to a species with the same molecular weight as the newly synthesized DNA‐binding protein, indicating that the modification of the protein is due to phosphorylation.
Transbilayer Distribution and Mobility of Phosphatidylcholine in Intact Erythrocyte Membranes
Authors: VAN MEER Gerrit , POORTHUIS Ben J. H. M. , WIRTZ Karel W. A. , OP DEN KAMP Jos A. F. , VAN DEENEN Laurens L. M.
Published: March 2005

The exchange of phosphatidylcholine between intact human or rat erythrocytes and rat liver microsomes was greatly stimulated by phosphatidylcholine‐specific exchange proteins from rat liver and beef liver. It was found, however, that compared to the exchange reaction between phospholipid vesicles and rat liver microsomes, much higher concentrations of exchange protein were required in the case of intact red blood cells and microsomes.

In human erythrocytes, 75% of the phosphatidylcholine was available for exchange within 2 h at 37°C. No additional exchange was observed during the next 2 h, indicating slow, if any, trans‐bilayer movement of the residual phosphatidylcholine.

In rat erythrocytes 50–60% of the phosphatidylcholine was readily available for the exchange proteins. The residual phosphatidylcholine was exchanged at a much lower rate with a half time for equilibration of 7 h.

These results confirm in an independent way the asymmetric distribution of phosphatidylcholine over the membrane of human and rat erythrocytes as well as the occurrence of a slow transbilayer movement of this lipid in rat erythrocytes.
31P Nuclear‐Magnetic‐Resonance Studies of Energy Metabolism in Tissue from the Marine Invertebrate Tapes watlingi
Authors: BARROW Kevin D. , JAMIESON Dana D. , NORTON Raymond S.
Published: March 2005

31P nuclear magnetic resonance spectra of tissue from the marine bivalve mollusc Tapes watlingi are presented. Spectra from foot muscle, adductor muscle and hearts contain resonances from the adenine nucleotides, inorganic phosphate and a phosphagen which is shown to be arginine phosphate. The intracellular pH of the tissue, measured from the chemical shift of the inorganic phosphate resonance, is 7.2, which is significantly lower than that of sea water, viz. 8.1. The importance of a correct choice of solution conditions for determining a correlation curve of chemical shift versus pH for tissue from marine organisms is emphasized. Spectra of perchloric acid extracts of muscle are used to determine levels of the major phosphorus‐containing metabolites in the tissue.

The time courses of changes in the phosphorus‐containing metabolites of foot and adductor muscle are described. Under anaerobic conditions the sequence of events is similar to that observed in vertebrate skeletal muscle, but the overall rate of decay is much slower, e.g. arginine phosphate is present in foot muscle for almost 20 h at its physiological temperature. Oxygenation of the incubation medium prolongs the survival time of the tissue. Treatment with iodoacetamide and sodium azide under anaerobic conditions reduces the lifetime and changes the pattern of decay such that ATP begins to decay before the phosphagen is consumed, and most of the phosphate accumulates in sugar phosphates, mainly fructose 1,6‐biphosphate.

The effect of low salinity stress is also examined. Spectra of muscle from animals exposed to 50% and 75% normal sea water for periods of about four weeks are essentially identical with those of normal animals. Furthermore, the decay of intact muscle incubated in 50% sea water is similar to that in normal sea water.
Bile Acid Synthesis in Isolated Rat Liver Cells
Authors: BOTHAM Kathleen M. , BECKETT Geoffrey J. , PERCY‐ROBB Iain W. , BOYD George S.
Published: March 2005

Bile acid synthesis in isolated rat hepatocytes was measured using radioimmunoassays for conjugated cholic and chenodeoxycholic acids. Synthesis of both bile acids was increased in hepatocytes from rats fed a diet containing the bile‐acid‐sequestering agent, cholestyramine, as compared to cells from rats given a pellet diet. When the cholestyramine‐fed rats were subjected to a reversed light cycle a further increase in bile acid synthesis was observed in the hepatocytes. Addition of 7α‐hydroxycholesterol at a concentration of 0.068 mM to the incubation medium of the hepatocytes stimulated 2–3‐fold the synthesis of both conjugated cholic and chenodeoxycholic acids. Conjugated chenodeoxycholic acid synthesis was also increased at concentrations of 0.14 and 0.68 mM 7α‐hydroxycholesterol, but conjugated cholic acid synthesis was inhibited at these levels. This suggests that high concentrations of 7α‐hydroxycholesterol may inhibit the 12α‐hydroxylation step in bile acid synthesis. Other oxygenated derivatives of cholesterol such as the 12α‐hydroxy, 25‐hydroxy, 26‐hydroxy and 7‐oxo derivatives did not stimulate bile acid synthesis in the liver cells. It is concluded that the increase in bile acid synthesis found in isolated hepatocytes on addition of 7α‐hydroxycholesterol to the incubation medium is specific for this compound, indicating that cholesterol‐7α‐hydroxylase is the rate‐limiting enzyme in the biosynthetic pathway from cholesterol to bile acids.
Messenger RNA Coding for Phenylalanine Ammonia‐Lyase
Authors: RAGG Hermann , HAHLBROCK Klaus
Published: March 2005

The mRNA coding for phenylalanine ammonia‐lyase was partially purified from irradiated cell suspension cultures of parsley (Petroselinum hortense). The product of cell‐free translation of the mRNA in a reticulocyte lysate was isolated by immunoprecipitation and compared with the native enzyme subunit. Evidence for the identity, or at least a great similarity, of both was provided by tryptic‐peptide and gel‐electrophoretic analyses.

Under partially denaturing conditions, phenylalanine ammonia‐lyase mRNA sedimented as a 20–21‐S molecule in a sucrose gradient and had an apparent molecular weight of about 1.05 × 106on a polyacrylamide gel. Approximately two‐thirds of the polynucleotide sequence of the mRNA were estimated to be required as coding sequence for the enzyme. We suggest that phenylalanine ammonia‐lyase mRNA is unlikely to code for more than one of three coordinately induced enzymes.
Ligand‐Linked Changes of Ultrasound Absorption of Hemoglobin
Authors: JÜRGENS Klaus D. , BAUMANN Rosemarie , RÖBBEL Heike
Published: March 2005

The acoustic absorption of hemoglobin solutions at 1.88 MHz has been studied in dependence of ligand binding and pH. In the physiological pH range the excess absorption of oxyhemoglobin is increased compared to deoxyhemoglobin. Evidence is presented that in human hemoglobin the additional absorption of oxyhemoglobin is caused by a proton transfer relaxation process involving the α chain N‐terminal α‐amino groups. This process cannot be observed in deoxyhemoglobin because of fixation of the N‐terminal α‐amino groups in a salt bridge. Further investigations of carbonmonoxy, nitrosyl, aquomethemoglobin and fluoromethemoglobin in the absence and presence of inositol hexakissulfate show that acoustic absorption measurements offer an additional tool to characterize in greater detail the different conformational states of hemoglobin.
Immunological Cross‐reactivity of Multiplication‐Stimulating Activity Polypeptides
Authors: MOSES Alan C. , NISSLEY S. Peter , SHORT Patricia A. , RECHLER Matthew M.
Published: March 2005

The immunoreactivity of the multiple species of multiplication‐stimulating activity (MSA) purified from medium conditioned by a rat liver cell line (BRL‐3A) has been examined. Antibodies were raised in rabbits following immunization with MSA II polypeptides. Subpopulations of antibodies were purified from one antiserum using DEAE‐cellulose chromatography. One antibody subpopulation recognized common antigenic determinants on MSA I and MSA II polypeptides; whereas a second antibody subpopulation recognized common determinants on MSA I, II, and III polypeptides. In a radioimmunoassay utilizing 125I‐MSA III‐2 and a purified antibody subpopulation, the human somatomedins (somatomedin A, insulin‐like growth factor I and II) showed weak, but significant cross‐reactivity: insulin‐like growth factor II was 10% as potent as MSA II. By contrast, somatomedin partially purified from rat serum, insulin, growth hormone, epidermal growth factor, nerve growth factor, and fibroblast growth factor, showed no reactivity in the radioimmunoassay.