bluuebunny/crossref_metadata_2025 huggingface dataset:
The Dolichol Pathway of Protein Glycosylation in Rat Liver
Authors: GODELAINE Danièle , BEAUFAY Henri , WIBO Maurice
Published: March 2005

Incorporation of mannose into endogenous lipid and protein acceptors of the rat liver endoplasmic reticulum was investigated by incubating rough microsomes with GDP‐[14C]mannose in the absence of detergent. Labeled dolichyl monophosphate mannose was readily formed from the endogenous dolichyl monophosphate. In contrast, the label was not detectable in dolichyl diphosphate oligosaccharides and it was incorporated into protein only to a small extent, except when preparations previously treated with pyrophosphate were incubated in the presence of UDP‐N‐ acetylglucosamine and GTP. Then, incorporation of label into such lipid and protein derivatives was considerably enhanced. GTP could not be substituted by other nucleotides. The requirement for UDP‐N‐acetylglucosamine, and other experimental evidence, demonstrate that mannosylated oligosaccharides which contain internal N‐acetylglucosamine units are entirely assembled on dolichyl diphosphate from the exogenous nucleotide sugars. The kinetics of mannose incorporation into dolichol‐linked oligosaccharides and into protein suggest that the protein acceptors are mainly mannosylated through transfer of oligosaccharides preassembled on dolichyl diphosphate. In heavy microsomes which have not been treated with PPi, or upon incubation in the absence of GTP, the amount of dolichyl phosphate which can accept N‐acetylglucosamine 1‐phosphate is less than that which can accept mannose. However, when PPi‐treated preparations are incubated with UDP‐N‐acetylglucosamine and GDP‐mannose in the presence of GTP, a larger amount of dolichyl phosphate is mobilized in the form of various saccharide derivatives of dolichyl diphosphate, and the amount of dolichyl monophosphate mannose is correspondingly decreased. It seems, therefore, that the membranes contain a particular pool of dolichyl monophosphate, which readily forms dolichyl monophosphate mannose, but reacts with UDP‐N‐acetylglucosamine only under appropriate conditions. This pool could act as precursor of the glycoside derivatives of dolichyl diphosphate which transfer their saccharide moiety to proteins.
Differential Effects of Two Interferon‐Induced Translational Inhibitors on Initiation of Protein Synthesis
Authors: CHERNAJOVSKY Yuti , KIMCHI Adi , SCHMIDT Azriel , ZILBERSTEIN Asher , REVEL Michel
Published: March 2005

At least two different mechanisms for the inhibition of mRNA translation operate in extracts of interferon‐treated L cells. One is mediated by an interferon‐induced protein kinase which, when activated by double‐stranded RNA and ATP, phosphorylates the small subunit of initiation factor eIF‐2. Addition of the purified interferon‐induced protein kinase to L cell extracts, strongly reduces the amount of methionyl‐tRNA bound to 40‐S ribosomal subunits. The second translational inhibition is due to the synthesis of (2′‐5′)oligo(adenylate) by interferon‐induced enzyme E. The oligonucleotide in turn activates a ribonuclease F constitutively present in L cells. Addition of the purified nuclease with its oligonucleotide activator to L cell extracts produces a strong decrease in polyribosome formation and an accumulation of initiation complex. These experiments differentiate the effects of the two interferon‐induced inhibitors on mRNA translation.
1H Nuclear‐Magnetic‐Resonance Studies of Porcine Lutropin and Its α and β Subunits
Authors: MAGHUIN‐ROGISTER Guy , DEGELAEN Jacques , ROBERTS Gordon C. K.
Published: March 2005

The titration curves of the histidine residues of porcine lutropin and its isolated α and β subunits have been determined by following the pH‐dependence of the imidazole C‐2 proton resonances. The isolated α subunit contains a buried histidine, whose C‐2 proton does not exchange with solvent, and which has the unusually low pK of 3.3. In the native hormone all the histidine residues have relatively normal pK values (between 5.7 and 6.2).

The four histidine C‐2 proton resonances have been assigned to specific residues in the amino‐acid sequence, by means of deuterium and tritium exchange experiments on the α subunit and its des(92–96) derivative. The histidine with a pK of 3.3 is identified as His‐α87.

The effects of pH on tyrosine and methyl proton resonances show that the titration of His‐87 in the isolated α subunit is accompanied by a significant conformational change which involves loosening of the protein structure but which is not a normal unfolding transition.

The role of conformational changes in the generation of biological activity by subunit association in the glycoprotein hormones is discussed.
Anaerobic Respiration and Energy Conservation inParacoccus denitrficans
Authors: MEIJER Emmo M. , VAN DER ZWAAN J. Wim , STOUTHAMER Adrian H. , WEVER Ronald
Published: March 2005

Electron paramagnetic resonance spectra at 8–60 K of NADH‐reduced membrane particles prepared fromParacoccus denitrificansgrown anaerobically with nitrate as terminal electron acceptor show the presence of iron‐sulfur centers 1–4 in the NADH‐ubiquinone segment of the respiratory chain. In addition resonance lines atg= 2.058,g= 1.953 andg= 1.88 are detectable in the spectra of succinate‐reduced membranes at 15 K, which are attributed to the iron‐sulfur‐containing nitrate reductase.

Sulphate‐limited growth under anaerobic conditions does not affect the iron‐sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain‐linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate‐limited growth ofP. denitrificansunder aerobic conditions [Eur. J. Biochem.(1977)81, 267–275].

Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain‐linked energy conservation observed during nitrate respiration [Arch. Microbiol.(1977)112, 17–23] can be explained by the uncoupling action of nitrite.
Inhibition of the Aliphatic Amidase from Pseudomonas aeruginosa by Urea and Related Compounds
Authors: GREGORIOU Mary , BROWN Paul R.
Published: March 2005

The time‐dependent inhibition of amidase from Pseudomonas aeruginosa strain AI 3 by urea, hydroxyurea and cyanate displayed saturation kinetics fitting a model for the reaction sequence in which formation of a complex in a reversible step was followed by an irreversible step. Altered amidases from mutant strains AIU 1N and OUCH 4, selected for their resistance to inhibition of growth by urea and hydroxyurea respectively, had altered kinetic constants for inhibition indicating reduced binding capacity for the inhibitors. The substrate acetamide protected AI 3 amidase against inhibition by urea, and altered Ki values for inhibition of the mutant amidases were paralleled by alterations in Km values for acetamide indicating that urea acted at the active site. Inhibition of AI 3 amidase involved the binding of one molecule of urea per molecule of enzyme. Urea inhibited amidase slowly regained activity at pH 7.2 through release of urea.
Synthesis of Chloroplast Ribonucleic Acid in Chlamydomonas reinhardtii Toluene‐Treated Cells
Authors: GUERTIN Michel , BELLEMARE Guy
Published: March 2005

Chlamydomonas reinhardtii cells treated with toluene at 0°C and 25°C incorporate ribonucleoside triphosphates (NTPs) into chloroplast RNA at 25°C and also at 35°C. The incorporation requires all four NTPs and Mg2+, and is completely inhibited by DNase, RNase, actinomycin D (40 μg/ml) and rifampicin (350 μg/ml). However, the incorporation is almost totally insensitive to both α‐amanitin and streptolydigin at 200 μg/ml.
Analysis of the Vicia faba Genome by Use of Restriction Endonucleases
Authors: KAINA Bernd , HINZ Rainer
Published: March 2005

DNA of the broad bean, Vicia faba, was cleaved by the restriction endonucleases endoR ·EcoRI, endoR ·HindIII, endoR ·HincII, endoR ·BamI, and endoR ·BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme‐specific pattern of bands composed of restriction fragments of 300 to more than 30000 base pairs in length.

Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species (∼ 75%).

In all digests, a class of high‐molecular‐weight restriction fragments of more than 30000 base pairs in length was observed which comprised 5–8% of the genome. It showed hybridization with highly repetitive DNA (c0t 2 × 10−2 M · s) and included a fraction (2–3% of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR ·EcoRI digests, the high‐molecular‐weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content: 61%) containing cleavage sites for the other enzymes used.
Kinetic Studies of the Interaction between MS2 Phage and F Pilus of Escherichia coli
Authors: DATE Takayasu
Published: March 2005

The kinetics of the binding reaction of MS2 phage to free F pili, which were highly purified from Escherichia coli, has been studied using a membrane filter assay. The rate of dissociation (kd) of the MS2‐phage–F‐pilus complex is very slow and follows first‐order kinetics with a half‐life of 4.2 h at 30 °C in the standard buffer. The dissociation rate is rather insensitive to temperature, but becomes more rapid at high ionic strength or at basic pH. In a 0.25 M ionic strength buffer, the half‐life of the complex is about 1.0 min. The rate of association is very fast and follows second‐order kinetics with the rate constant for association (ka) being 8 × 107 M−1 s−1 at 30°C in the standard buffer. The rate of association is almost insensitive to ionic strength but slightly sensitive to pH or temperature. Monovalent cations can also promote the binding reaction as well as divalent cations but the complex formed with monovalent cation is unstable. A study of the kinetics of dissociation suggests that there are two types of interaction between MS2 phage and F pilus; one is a strong interaction formed with divalent cations and the other is a weak one formed with monovalent cations. The physical nature of the bonds involved in the former and the latter seems to be mainly electrostatic and non‐electrostatic respectively. The mechanism of the binding reaction is discussed.
The Complete Amino‐Acid Sequence of the Sweet Protein Thaumatin I
Authors: IYENGAR Ramanuja B. , SMITS Petrus , VAN DER OUDERAA Frans , VAN DER WEL Hendrik , VAN BROUWERSHAVEN Johan , RAVESTEIN Peter , RICHTERS Gerard , VAN WASSENAAR Pieter D.
Published: March 2005

The primary structure of the sweet‐tasting protein thaumatin has been elucidated. The protein consists of a single polypeptide chain of 207 residues. The sequence of the N‐terminal part of the chain was determined by sequenator analysis. As the protein contains only one methionine residue, it was possible to deduce the N‐terminal sequence of the C‐terminal cyanogen bromide fragment by automatic sequencing of the cyanogens‐bromide‐cleaved, succinylated protein. To arrive at the sequence of the whole protein tryptic and Staphylococcus protease peptides, together with chymotryptic peptides and a 2‐(2‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine (BNPS‐skatole) fragment were also sequenced.

Comparing the amino acid sequence of thaumatin with that of the other sweet‐tasting protein, monellin, we have located five sets of identical tripeptides. Since immunological cross‐reactivity of thaumatin antibodies with monellin has recently been described, one or more of these tripeptides might be part of a common antibody recombination site and possibly be involved in the interaction with the sweet‐taste receptor.
Purification of Sea‐Urchin RNA Polymerase II
Authors: BALLARIO Paola , Di MAURO Ernesto , GIULIANI Clara , PEDONE Francesco
Published: March 2005

The purification of RNA polymerase II from gastrulae of Paracentrotus lividus is described. The enzyme obtained is homogeneous as judged by electrophoresis under non‐denaturing conditions. It is able to transcribe both native high‐Mr P. lividus DNA and Psammechinus miliaris h22 histone DNA, although single‐stranded and nicked DNAs are better templates. P. lividus RNA polymerase II forms with homologous native DNA stable binary complexes that are able to initiate RNA chains after exposure to heparin. Heparin‐resistant complexes do not form on nicks of DNA molecules.

Sensitivity of sea‐urchin RNA polymerase II to rifamycin derivatives and α‐amanitin has been determined.
Studies on Ribosomal Proteins in the Cellular Slime Mold Dictyostelium discoideum
Authors: RAMAGOPAL Subbanaidu , ENNIS Herbert L.
Published: March 2005

This study is concerned with the identification and subunit localization of ribosomal proteins in Dictyostelium discoideum. The characterization is based on the resolution of ribosomal proteins by various methods of electrophoresis. 34 and 42 unique proteins were identified in the 40‐S and 60‐S ribosomal subunits respectively. The total mass of proteins in the 40‐S subunit was 746100 daltons and 981900 daltons in the 60‐S subunit. The molecular weights of individual proteins in the 40‐S subunit ranged from 13200 to 40900 with a number‐average molecular weight of 21900. The molecular weight range for the 60‐S subunit was 13800 ‐ 51100 with a number‐average molecular weight of 23400. The 80‐S ribosome contained 78 proteins, two of which were lost upon its dissociation into subunits. All the proteins of the 40‐S and 60‐S subunits could be identified individually in a 80‐S map as well as in unfractionated proteins from whole cells. Purification of ribosomes in high‐ionic‐strength buffers resulted in non‐specific ioss of the various proteins from the 40‐S and 60‐S subunits. In addition, the undissociated ribosomes contained about 10 acidic proteins in the molecular weight range 50000‐100000, which were retained after washing the ribosomes in highsalt buffers. They were found in polysomes, run‐off ribosomes and could also be identified in the 40‐S subunit after dissociation.
Isolation and Characterization of an Ornithine‐Containing Lipid from Paracoccus denitrificans
Authors: THIELE Otto Wolfgang , BISWAS Clare Jamie , HUNNEMAN Donald H.
Published: March 2005

The isolation and characterization of an ornithine‐containing lipid from various strains of Paracoccus denitrificans (grown heteretrophically and autotrophically) is reported. The structure of this aminolipid was found to be H2N ‐CH2 ‐(CH2)2 ‐CH[NH ‐CO ‐CH2 ‐CH(O ‐CO ‐R1) ‐R2]‐CO2H, where R1 predominantly represents the residue of octadec‐11‐enoic acid, R2 the residue of a 3‐hydroxyeicos‐13‐enoic acid.

In addition, the major phospholipids (phosphatidylcholine, phosphatidylethanolamine, phos‐phatidylglycerol) were isolated and characterized.
Sodium‐23 Nuclear Magnetic Resonance as an Indicator of Sodium Binding to Troponin C and Tryptic Fragments, in Relation to Calcium Content and Attendant Conformational Changes
Authors: DELVILLE Alfred , GRANDJEAN Jean , LASZLO Pierre , GERDAY Charles , GRABAREK Zenon , DRABIKOWSKI Witold
Published: March 2005

The relaxation rate enhancements of the 23Na nuclei for NaHCO3 solutions of troponin C and its tryptic peptides TR‐1 and TR‐2 indicate true binding of Na+ ions to these biomolecules. The low‐affinity sites I and II of TR‐1 and troponin C are the sites of competitive Na+/Ca2+ binding, below one calcium ion per molecule, with log KNa∼ 2. At low calcium content Na+ ions bind to TR‐2 and to troponin C non‐competitively with Ca2+ ions; binding of Ca2+ ions to the high‐affinity sites III and IV allosterically affects the binding of the Na+ ions: even when sites I and II, located on TR‐1 or sites I, II. III, IV of troponin C, are saturated with Ca2+ ions, Na+ ions continue to bind weakly at secondary binding sites.
Interaction of Escherichia coli EF‐Tu · GTP and EF‐Tu · GDP with Analogues of the 3′ Terminus of Aminoacyl‐tRNA
Authors: JONÁK Jir̆i , SMRT Jir̆i , HOLÝ Antonín , RYCHLÍK Ivan
Published: March 2005

The affinity of chemically synthetized analogues of the 3′ terminus of phenylalanyl‐tRNA and leucyl‐tRNA for the aminoacyl‐tRNA binding site of Escherichia coli elongation factor‐Tu · GTP complex (EF‐Tu · GTP) increases in the order A(Phe)°C‐A(Leu)<pA(Phe)≤C‐A(Phe)<amino‐acyl‐tRNA [2′(3′)‐O‐l‐phenylalanyladenosine<cytidylyl‐(3′≤5′)‐2′(3′)‐O‐l‐leucyladenosine≤2′(3′)‐0‐l‐phenylalanyladenosine 5′‐phosphate<cytidylyl‐(3′→ 5′)‐2′(3′)‐0‐l‐phenylalanyladenosine<aminoacyl‐tRNA], as we revealed using the l‐l‐tosylamido‐2‐phenylethyl chloromethyl ketone assay [(1979) FEES Lett. 93, 329‐332]. By contrast, C‐A(d‐Phe), C‐A(fPhe), and an equimolar mixture of C‐A+phenylalanyl methyl ester are at least twenty times less active than C‐A(Phe) and do not differ from C‐A alone; puromycin is almost inactive. The affinity of A(Phe), pA(Phe), C‐A(Phe) and C‐A(Leu) (just like the affinity of the intact molecule of aminoacyl‐tRNA) for EF‐Tu · GDP is substantially lower than for EF‐Tu · GTP. However, with puromycin, C‐A, C‐A(o‐Phe), C‐A(fPhe) and an equimolar mixture of C‐A+phenylalanvl methyl ester this difference between EF‐Tu · GTP and EF‐Tu · GDP is no longer observed or is almost insignificant.

These results indicate that the binding site for the 3′terminus of aminoacyi‐tRNA in EF‐Tu · GTP is intimately involved in the fundamental property of the factor to discriminate between aminoacyl‐tRNA, peptidyl‐tRNA and tRNA. The presence of the undisturbed 2′(3′)‐O‐l‐aminoacyladenosine structure at the 3′ end of respective ligands appears to be a prerequisite for the recognition of the ligand by the aminoacyl‐tRNA binding site of EF‐Tu · GTP. In addition, this site apparently also contains regions which interact with the phosphate residue and with cytidine of the 3′‐terminal C‐A sequence of aminoacyl‐tRNA and can contribute significantly to the stability of the interaction. The results support the idea of the key role of the 3′‐terminal C‐C‐A structure of tRNA as ‘general ligand’ in protein synthesis.

Finally, our results indicate that the occupation of the guanosine nucleotide binding site of EF‐Tu either by GTP or GDP could control the affinity ofthe factor for the whole molecule of aminoacyl‐tRNA simply by modulating the affinity of EF‐Tu just for the 3′ terminus of the poly‐nucleotide.
Transfer of Radioactive Materials from Radioactively Labeled Conditioned Medium to Fixed Cells
Authors: SAKIYAMA Hisako
Published: March 2005

Transfer of radioactive materials to glutaraldehyde‐fixed Ni12Cl cells was observed when fixed cells were incubated in conditioned media of Ni12Cl cells cultured with various radioactive precursors such as glucosamine, fucose or leucine. Radioactivities were not transferred to fixed cells when the conditioned medium was boiled. The transfer decreased drastically by treatments of the medium with trypsin or detergents (dodecylsulfate or deoxycholate). Efficient transfer occurred only at the physiological temperature. No radioactivity was transferred to fixed cells if conditioned media of nonlabeled cells and radioactive precursors including various nucleotide sugars were incubated with fixed cells.

The transferring activity in a conditioned medium did not decrease by the dialysis or the centrifugation at 105000 ×g for 2 h, and remained in the supernatant. The activity migrated into the sucrose gradient.

Materials transferred to fixed cells were not solubilized by detergents but were released by the treatment with trypsin, indicating transferred materials were covalently associated with fixed cells.

Those results suggest that conditioned media thus obtained contain both substrate(s) and enzyme(s) which fixed labels to fixed cells.
Origin of the Selectivity of α‐Dicarbonyl Reagents for Arginyl Residues of Anion‐Binding Sites
Authors: PATTHY László , THÉSZ János
Published: March 2005

Factors affecting the reactivity of arginyl side‐chains of proteins toward α‐dicarbonyl reagents have been considered.

The reaction of the guanidino group with different α‐dicarbonyl compounds is shown to proceed by a common pathway, the rate‐limiting step being essentially identical with an amine–carbonyl addition reaction. We found that the protonated guanidino group is the main reacting species in acidic solutions; at neutral–alkaline pH, however, reaction of the unprotonated form is predominant. Under the conditions usually employed for the modification of enzymes only the pathway utilizing the unprotonated form of arginine is of importance, the reactivity of individual arginyl side‐chains toward α‐dicarbonyls is primarily determined by the pka value of the guanidinium groups.

It is proposed that the pKa value of arginyl residues of anion binding sites is lower than that of other arginyl residues, due to the strong positive electric potential of the anion binding sites. The lower pKa of arginine leads to its hyperreactivity toward the different α‐dicarbonyl reagents, providing an explanation for the general observation that α‐dicarbonyl reagents are remarkably selective for arginyl residues involved in anion binding.

The various predictions of our proposition are considered and tested experimentally on the arginine residue of the high‐affinity anion binding site of rabbit muscle aldolase.
Polyamines Stimulate Suppression of Amber Termination Codons in vitro by Normal tRNAs
Authors: MORCH Marie‐Dominique , BENICOURT Claude
Published: March 2005

Polyamines, such as spermine and spermidine, are able to stimulate reading of amber termination codons on viral messenger RNAs in vitro. This phenomenon is not due to an overall increase of error frequency during translation, but to a specific effect on a normal tRNA that is present in various eukaryotic cell preparations. The enhancement of reading of termination codons by normal tRNAs should be of major importance for the expression of specific genes in eukaryotic cells.
Tyrosine Residues in the C‐Terminal Domain of the Elongation Factor G Are Essential for Its Interaction with the Ribosome
Authors: ALAKHOV Yuly B. , ZALITE Indulis K. , KASHPAROV Ivan A.
Published: March 2005

Chemical modification of the elongation factor G (EF‐G) with tetranitromethane and iodine has been studied. It has been shown by spectrophotometric titration that EF‐G contains two exposed tyrosine residues, one of which has an unusually low pK value for a phenol hydroxyl group at pH 8.5. Modification of one tyrosine residue with either tetranitromethane or iodine results in a 70‐80% loss of EF‐G activity in all ribosome‐dependent reactions. Modification of three or four residues inhibits 90‐100 % of activity. Binding of EF‐G with the 70‐S ribosome and 50‐S subunit is equally effective for protection of tyrosine residues against modification. The rate of EF‐G modification with tetranitromethane is considerably higher in the presence of guanyl nucleotides than for free EF‐G. The modified residues are located in the C‐terminal domain of EF‐G and are presumably contained in one of the sites of EF‐G interaction with the ribosome.
Studies by Small‐Angle X‐Ray Scattering of the Quaternary Structure of the 24‐S Component of the Haemocyanin of Astacus leptodactylus in Solution
Authors: PILZ Ingrid , GORAL Karin , HOYLAERTS Marc , WITTERS Raphaël , LONTIE René
Published: March 2005

The haemocyanin of Astacus leptodactylus was studied in several buffers at three pH values. The best stability and lowest mean deviation on repeating the measurements were found at pH 7.2 in a Tris‐HCl buffer. The following molecular parameters were determined: radius of gyration 6.90 nm, radius of gyration of the cross‐section 3.87 nm, maximum dimension 21.5 nm, relative molecular mass 854000, volume 1440 nm3, hydration 0.27 g H2O/g protein. The theoretical scattering curves of a large number of models were calculated to find one fitting these data and the experimental scattering curve. The model with the best agreement was compared with an electron micrograph.
Relationships between α and β Secondary Structures and Amino‐Acid Pseudosymmetrical Arrangements
Authors: DELHAISE Philippe , WUILMART Christian , URBAIN Jacques
Published: March 2005

A total of 51 polypeptides of known amino acid sequence and secondary structure have been screened for the presence of symmetrical arrangements of amino acids. Similarity between amino acids was derived by using a genetic test (minimum mutation distance) or a structural test (relative frequencies of amino acids substitutions in families of related proteins). It is shown that the frequency of proteins displaying symmetrical arrangements of amino acids is slightly higher than predicted by chance.

In contrast, when the analysis is restricted to protein subregions displaying identical types of secondary structure, the frequency of proteins in which the α and β subregions exhibit symmetrical arrangements of amino acids is significantly higher than predicted by chance. On the other hand, it is observed that more discriminatory results are always obtained when the structural test is used as a criterion for amino acid similarity.

These data suggest that symmetrical arrangements of amino acids could result from structural constraints imposed either by the α and β secondary structures. It is postulated that the regular alternation in hydrophobicity which is generally observed in the amino acid sub‐sequences displaying α or β secondary structures may be responsible for the occurrence of symmetrical arrangements of amino acids.
The Reaction of Bovine Pancreatic Ribonuclease A with 6‐Chloropurineriboside 5′‐Monophosphate
Authors: PARÉS Xavier , LLORENS Rafel , ARÚS Carles , CUCHILLO Claudi M.
Published: March 2005

The chemical modification of bovine pancreatic ribonuclease A by 6‐chloropurine 9‐β‐d‐ribo‐furanosyl 5′‐monophosphate was studied under several reaction conditions. The reaction, at pH 7.3, 40°C and a nucleotide: enzyme molar ratio of 60, showed a high degree of specificity in comparison to those corresponding to the base or the nucleoside. The main derivative was isolated by means of CM‐cellulose chromatography. Subtilisin cleavage of this derivative showed that the substitution had taken place in the S‐peptide moiety. Tryptic digestion of the S‐peptide indicated that a lysine residue had been modified. Enzymatic and physico‐chemical considerations showed that the actual site of reaction was the α‐amino group of Lys‐1. The structural and kinetic properties of the derivative are consistent with the existence of a phosphate‐binding sub‐site near the N‐terminal region of the enzyme.
A Proton‐Magnetic‐Resonance Study of N‐Trifluoroacetyl‐l‐alanyl‐l‐phenylalaninal Binding to α‐Chymotrypsin
Authors: WYETH Paul , SHARMA Ram P. , AKHTAR Muhammad
Published: March 2005

Cross‐saturation NMR studies have shown that the dipeptide aldehyde N‐trifluoroacetyl‐l‐alanyl‐l‐phenylalaninal forms a hemiacetal complex with α‐chymotrypsin. The free aldehyde resonance was seen to broaden upon addition of α‐chymotrypsin and a detailed analysis has identified the enzyme‐bound hemiacetal as the species which was in slow exchange with the free aldehyde and so gave rise to the perturbation. The line broadening was found to be dependent on p2H and this behaviour could be adequately described by the ionisation of a single group on the free enzyme (pK2Ha 7.9) with the alkaline form being required for hemiacetal formation.

The pH dependence of α‐chymotrypsin‐induced catalysis around neutral pH correlates well with the observed p2H dependence of hemiacetal formation, indicating that it is an intermediate in the catalytic mechanism.
Purification and Some Properties of Aldehyde Rcductases from Pig Liver
Authors: BRANLANT Guy , BIELLMANN Jean‐François
Published: March 2005

Two aldehyde reductases (EC 1. 1. 1. 2), I and II, have been isolated from pig liver. Both are monomeric (Mr∼ 35000 and NADPH‐dependent. Their activity is inhibited by barbiturates. The enzymes reduce essentially aromatic aldehydes, with a preference for those bearing an electron‐withdrawing group in the para position. Substrates with a carboxyl group are specially good substrates for reductase I. This may indicate the presence of a positively charged group in the substrate binding site.

The binding of NADPH to reductase I causes a red shift of the coenzyme absorption; this shift characteristic of B‐stereospecific dehydrogenases. Nevertheless, this is not confirmed by the stereochemical study with labelled NADPH. The pro‐R hydrogen of NADPH is transferred to the re face of the aldehyde. The stereochemical course of reductase I is identical to that of liver alcohol dehydrogenase, but the two enzymes differ by the absence of Zn and of reactive thiol in reductase I, and by the action of pyrazole on the activity. Considerable differences in substrate specificity and immunological properties have been found between reductase I and II but reductases I from liver of different species have some relationship. Reductase I from pig brain and pig kidney seem to be identical to reductase I from pig liver.
Circular Dichroism of Elapidae Protein Toxins
Authors: DRAKE Alex F. , DUFTON Mark J. , HIDER Robert C.
Published: March 2005

The effect of temperature variation on the circular dichroic spectra of four Elapid snake toxins (one short neurotoxin, two long neurotoxins and one cytotoxin) was determined and the results compared.

The three neurotoxins have in common a delicately balanced conformation which can be perturbed independently of the predominant secondary structuring. Despite the many differences in sequence between long and short neurotoxins, the nature of this balance seems to be the same in both types and it is considered to involve an equilibrium between two defied conformers. Such properties could not be discerned in the cytotoxin studied, but related work suggests that they could nevertheless be present in some cases.

In terms of global structure, long neurotoxins may have a greater proportion of ‘random chain’ inherent than short neurotoxins, the latter having extensive β‐sheet.

The important feature of all toxins is deemed to be a conformational freedom defined by the secondary structuring and characteristic disulphide bridge formation. Possible relevance to toxicity of such a property is discussed.
Mitochondrial ATP: AMP Phosphotransferase from Beef Heart: Purification and Properties
Authors: TOMASSELLI Alfredo G. , NODA Lafayette H.
Published: March 2005

ATP: AMP phosphotransferase has been purified 186‐fold from beef heart mitochondria in an overall yield of 30%. The purified enzyme, Mr 31500, has a specific activity of 800 U/mg and was found to be a homogeneous protein by constancy of specific activity across the elution peak following passage through an Affi‐Gel Blue agarose column and by dodecylsulfate gel electrophoresis. Isoelectrofocusing shows a minor peak of activity that arises again when the major peak is re‐focused. The pI of the enzyme is 9.30 and the optimum pH of activity is 5.8 in the forward reaction (formation of ADP) and 8.0 in the reverse reaction. The enzyme requires the presence of a divalent metal ion, namely Mg2+, Co2+, Ca2+, Mn2+, Ni2+ and Zn2+ in decreasing order of activity and is influenced by NaCl. It shows rigorous specificity for AMP (dAMP) as phosphate acceptor but is rather unspecific for the triphosphate donor since, UTP, CTP, ITP and GTP may substitute for ATP (dATP). ATP and MgATP (dATP was not tested) bind to the enzyme with dissociation constants of 0.122 and 0.132 mM respectively, while AMP (dAMP was not tested), UTP, CTP, ITP and GTP binding could not be detected by the dialysis equilibrium technique used. Amino acid analysis gave the following composition as residues: 26 aspartic acid or asparagine, 14 threonine, 17 serine, 27 glutamic acid or glutamine, 24 proline, 19 glycine, 25 alanine, 17 valine, 8 methionine, 14 isoleucine, 31 leucine, 5 tyrosine, 9 phenylalanine, 6 histidine, 25 lysine, 15 arginine, 4 half cystine (no free sulfhydryls) and no tryptophan, giving a total of 286 residues.

The purified mitochondrial adenylate kinase is not a contaminating heart cytosolic enzyme. ATP:AMP phosphotransferase was extracted from beef heart cytosol depleted of mitochondria and brought to homogeneity. Determinations of pI, Mr and amino acid composition showed that the heart mitochondrial and cytosolic adenylate kinases are different isozymes.
Nuclear‐Magnetic‐Resonance Studies of Ferrocytochrome c
Authors: MOORE Geoffrey R. , WILLIAMS Robert J. P.
Published: March 2005

The pH dependence and the temperature dependence of the nuclear magnetic resonance spectrum of horse ferrocytochrome c are described. This protein is very stable; it maintains an ordered structure over the pH range 4 to 12 at 25 °C and over the temperature range 4°C to 97 °C at pH 7.0.

The dynamic characteristics of the conformation of ferrocytochrome c were investigated. Particular emphasis was laid on the aromatic resonances and resonances of methyl groups shifted far upheld. Tyr‐48 and Phe‐46 were found to be relatively immobile whilst a region of the protein close to Ile‐57 was found to be relatively flexible.
The Solution Structures of Tuna and Horse Cytochromes c
Authors: MOORE Geoffrey R. , WILLIAMS Robert J. P.
Published: March 2005

The nuclear magnetic resonance spectra of tuna ferricytochrome c and tuna ferrocytochrome c are described. Resonance assignments are made using NMR double‐resonance techniques. A comparison of the NMR data for tuna cytochrome c with the previously reported data for horse cytochrome c shows that the proteins have virtually identical main‐chain folds. Three regions of local conformational differences have been distinguished.
Interactions of Elongation Factor 2 (EF‐2) with Guanine Nucleotides and Ribosomes
Authors: NURTEN Rüstem , BERMEK Engin
Published: March 2005

Interactions of rat liver elongation factor 2 (EF‐2) with guanine nucleotides and ribosomes were studied by equilibrium dialysis and sedimentation methods. GDP (Kd= 0.5 μM) or GDP‐Mg2+ (Kd=1.57 μM) displayed a higher affinity in the formation of a binary complex with EF‐2 than GTP (Kd= 2.68 μM), GTP‐Mg2+ (Kd= 2.77 μM), or guanosine 5′‐[β,γ‐methylene]triphosphate (GuoPP[CH2]P) (Kd= 24.0 μM).

NaIO4‐oxidized guanine nucleotides (oGDP) (Kd= 38 μM) and oxidized/reduced guanine nucleotides (orGDP) (Kd= 27 μM) had lower affinites to the binding site on EF‐2 than those of GDP or GTP. However, the binding of oGDP, oGTP or oGuoPP[CH2]P to EF‐2 resulted in the formation of a stable product which could be recovered by the nitrocellulose filter technique or by sodium dodecyl sulfate/polyacrylamide gel electrophoresis.

In the presence of ribosomes and EF‐2 the formation of a new binding site (or a different conformation of the binding site) with a higher affinity for GuoPP[CH2]P‐Mg2+ (Kd= 0.26 μM) than for GDP‐Mg2+ (Kd= 9.3 μM) became apparent. The presence of ribosomes thus appeared to favor the formation of a complex involving guanosine triphosphates. Adenosine diphosphate ribosylated EF‐2 (ADP‐Rib‐EF‐2) in its turn could bind to the ribosome with high affinity even without guanosine nucleotides (Kd= 0.18 μM). GuoPP[CH2]P increased to some extent the affinity of ADP‐Rib‐EF‐2 for its ribosomal binding site (Kd= 0.05 μM).
The Structure of Cucurbitin: Subunit Symmetry and Organization in situ
Authors: COLMAN Peter M. , SUZUKI Eikichi , VAN DONKELAAR Albertus
Published: March 2005

The low‐resolution (2 nm) subunit symmetry of cucurbitin, the crystalline seed storage globulin of cucurbits, has been determined by X‐ray diffraction. The wet crystals belong to the cubic space group F23 and there are 4 molecules per unit cell. The molecules therefore possess point‐group symmetry 23 and contain 12 structural units which at this resolution are indistinguishable. On drying, the crystal lattice dimension shrinks from 13.6 nm to 12.4 nm with no apparent change in symmetry. Diffraction patterns of small crystals spun into a pellet, and sections of dry and wet native seed indicate that in situ the protein is organised in microcrystals of the same unit cell and symmetry.

Edestin, the crystalline storage globulin from cannabis, and a crystalline globulin from tobacco seed both have the same crystal lattice as cucurbitin and, very likely, the same subunit symmetry.
Cell‐Free Translation of Messenger RNAs from Adult and Fetal Human Muscle
Authors: KAHN Axel , COTTREAU Dominique , DAEGELEN Dominique , DREYFUS Jean‐Claude
Published: March 2005

Using a procedure of ethanol precipitation in concentrated guanidine · HCl solutions followed by chloroform/isoamylic alcohol extraction and washing in 3 M sodium acetate, we isolated high‐molecular‐weight cellular RNA from human fetal and adult skeletal muscle. About 500 μ RNA were obtained/g of fetal muscle and 50 μ RNA g of adult muscle. Both RNA preparations were efficiently translated in a cell‐free reticulocyte lysate system and directed synthesis of various polypeptides, one of them of Mr 200000 probably corresponding to myosin heavy chains. Dodecylsulphate/polyacrylamide gel electrophoretic pattern of polypeptides neosynthesized using either fetal or adult RNA exhibited several differences.

Three neosynthesized cytosolic muscle enzymes were purified from the translation mixtures using a micromethod of immunoaffinity chromatography; specificity of the neosynthesized polypeptides purified according to this procedure was checked by immunological competition with the corresponding unlabeled pure muscle enzymes.

Successful cell‐free translation of RNA from adult skeletal muscle and purification of neosynthesized human enzymes are reported for the first time. These methods, indispensable for further studies on human adult muscle gene expression, could also shed light on the mechanism of some inherited molecular diseases and tumoral or dystrophic processes.
Turnover of Cellular Glutathione in Isolated Rat‐Kidney Cells
Authors: MOLDÉUS Peter , ORMSTAD Kari , REED Donald J.
Published: March 2005

Turnover of cellular glutathione in isolated rat kidney cells was studied using cystine or methionine as sulfur donor. In the absence of any sulfur donor a continuous decrease of intracellular reduced glutathione (GSH) during incubation of the cells was observed. This decrease was abolished in the presence of cystine and, as indicated by incorporation of 35S, there was also a rapid synthesis of GSH. In the presence of a γ‐glutamyltransferase inhibitor, the synthesis of intracellular GSH was accompanied by an accumulation of extracellular cysteineglutathione mixed disulfide whereas only minor amounts of GSH and glutathione disulfide could be detected. The intracellular levels of both the cysteine‐glutathione and blutathione disulfides were at all time points very low.

Even though the uptake of cystine was rapid and not rate‐limiting for GSH synthesis, almost no cystine could be detected intracellularly. An increasing intracellular cysteine concentration was however observed, indicating a rapid reduction of cystine.

In contrast to cystine, methionine did not protect from the loss of intracellular GSH and only a low rate of incorporation of 35S into GSH was observed. Methionine was rapidly taken up into the cells but was apparently converted to cysteine only to a very limited extent. This is most likely due to a low activity of the enzyme cystathionase since neither homocysteine nor cystathionine was very effective in supporting GSH synthesis.
On the Molecular and Submolecular Structure of the Semiquinone Cations of Alloxazines and Isoalloxazines as Revealed by Electron‐Paramagnetic‐Resonance Spectroscopy
Authors: MÜLLER Franz , GRANDE Hans J. , HARDING Leonard J. , DUNHAM William R. , VISSER Antonie J. W. G. , REINDERS Jan H. , HEMMERICH Peter , EHRENBERG Anders
Published: March 2005

The thermodynamically stable and, therefore, analytically most important alloxazine and isoalloxazine radical cations have been studied in detail by electron paramagnetic resonance (EPR) spectroscopy. Isotopic and chemical substitutions have been made as in earlier studies with the less stable neutral and anionic species. The experimental spectra have been calculated with the aid of a more sophisticated computer‐simulation program than previously used. Excellent fits were obtained only when all of the following atoms were taken into account in the hyperfine coupling scheme: N‐5 H. N‐10 H or CH3. C‐6 H, C‐7 H, C‐8 H or CH3 and C‐9 H. An additional but small coupling constant was required for the fit. This latter coupling constant is assigned to the nitrogen atom(s) of the pyrimidine subnucleus of (iso)alloxazine radical cations. The EPR‐active proton is attached to N‐5 as we also found for the neutral flavosemiquinone.

The alloxazine and isoalloxazine radical cations exhibit an identical hyperfine coupling scheme but differ especially in the pyrazine nucleus with respect to the spin density distribution. This suggests that the geometrical structure of the two kinds of radicals is somewhat different. The highest spin density is, however, located at N‐5 of (iso)alloxazine as has been found for the other flavosemiquinone species.

The hyperfine coupling constants are interpreted in terms of spin densities and comparison is made with the most recently available quantum chemical calculations. All monomeric flavosemiquinone species are compared with each other and their differences in the submolecular structure are discussed briefly.
Asymmetric and Symmetric Membrane Reconstitution by Detergent Elimination
Authors: HELENIUS Ari , SARVAS Matti , SIMONS Kai
Published: March 2005

The dissociation and reconstitution of the Semliki Forest virus membrane using the nonionic detergent octyl β‐d‐glucoside was studied by sucrose density gradient centrifugation. The dissociation occurred in three stages: lysis at a free equilibrium octyl glucoside concentration of 14–18 mM, solubilization at 18–20 mM, and delipidation of the spike glycoproteins at the critical micellar concentration (22 mM) or higher. After solubilization the spike glycoproteins were present as soluble complexes with sedimentation coefficients of 19 S and 6 S. The 6‐S form probably corresponded to a glycoprotein monomer complexed to detergent and the 19‐S form consisted of oligomeric detergent‐protein complexes. The two forms were in slow equilibrium with each other.

When the soluble spike protein complexes and egg lecithin solubilized with octyl glucoside were mixed and the octyl glucoside concentration lowered either by dialysis or by dilution, reconstitution occurred. Three types of products were obtained: vesicles with 30% of the spike protein facing inwards and 70% facing outwards, vesicles with virtually all (95%) of the spike proteins pointing outwards, and small protein‐rich soluble aggregates [Helenius et al. (1977) J. Cell Biol. 75, 866]. It was demnnorated that during reconstitution the symmetric vesicles were formed at 19 mM free equilibrium octyl glucoside by the association of the 6‐S protein complexes with the phospholipids and the asymmetric vesicles were formed at 10–16 mM octyl glucoside when the 19‐S complexes associated with the lipids. Asymmetric membrane vesicles were also obtained when membrane penicillinase from Bacillus licheniformis was reconstituted with egg lecithin using octyl glucoside. It could be shown that the penicillinase was oligomeric at the octyl glucoside concentration where the reconstitution occurred.

The results demonstrate that different mechanisms of reconstitution give rise to the symmetric and the asymmetric vesicles. The critical factor in determining the mechanism is the state of aggregation of the proteins at the octyl glucoside concentration where membranes begin to form from the solubilized lipids.
The Amino‐Acid Sequence of the α Subunit in Bovine Brain S‐100a Protein
Authors: ISOBE Toshiaki , OKUYAMA Tsuneo
Published: March 2005

The brain specific S‐100 protein is a mixture of two components S‐100a and S‐100b with a subunit composition αβ or β2 respectively. The amino acid sequence of the β subunit has been previously determined. This paper presents the sequence of the α subunit in the S‐100a protein. The α subunit consists of 93 amino‐acid residues and has a relative molecular mass of 10400. The sequence shows extensive homology (58%) with that of the β‐subunit and shares an apparent calcium binding site in the C‐terminal half of the molecule, suggesting a close evolutionary relationship between these subunits.
Patterns of Product Inhibition of a Bifunctional Dehydrogenase; l‐Histidinol : NAD+ Oxidoreductase
Authors: BÜRGER Erich , GÖRISCH Helmut
Published: March 2005

The steady‐state kinetic patterns of the bifunctional enzyme histidinol dehydrogenase from Salmonella typhimurium (EC 1.1.1.23) are compatible with a bi‐uni uni‐bi ping‐gong mechanism. Studies of product inhibition make it possible to determine the sequence of substrate binding and product dissociation. Histidinol binds first to the enzyme, followed by the binding of NAD+; histidine is the last product to dissociate from histidinol dehydrogenase. Five of the ten kinetic constants defined are determined from linear intercept and slope replots: Km for histidinol was found to be 16 ± 3 μM and for NAD+ 1 ± 0.3 mM; K2 for NAD+ was determined to be 0.8 ± 0.4 mM and K3 for NADH to be 0.3 ± 0.07 mM. K1 for histidine was found to be 2.1 ± 0.5 mM.
Thionins: Plant Peptides that Modify Membrane Permeability in Cultured Mammalian Cells
Authors: CARRASCO Luis , VÁZQUEZ David , HERNÁNDEZ‐LUCAS Carlos , CARBONERO Pilar , GARCÍA‐OLMEDO Francisco
Published: March 2005

Thionins, which are high‐sulphur polypeptides present in the endosperm of wheat and related species, have been found to prevent growth and to inhibit macromolecular synthesis in cultured mammalian cells. Baby hamster kidney (BHK) cells were markedly more sensitive to thionins than the other cell lines tested (monkey CV1, mouse L, human HeLa). A thionin concentration of 5 μ/ml (1 μM) completely blocked translation in BHK cells. It was later found that omission of both calcium and magnesium ions from the medium strongly enhanced the inhibitory effects of thionins (BHK cells, 80% inhibition, 0.5 μ/ml). Several lines of evidence indicate that thionins might act at the membrane level. Indeed, both the 86Rb+ content and the nucleotide pool of BHK cells were drastically decreased at thionin concentrations that inhibited translation. In addition, thionin concentrations that did not affect macromolecular synthesis in these cells, allowed inhibition of translation by antibiotics, such as hygromycin Bthat are not able to cross the cell plasma membrane by themselves. Our results suggest that the inhibition of protein, RNA and DNA synthesis in BHK cells might be a consequence of membrane leakiness induced by thionin treatment. In this respect, particularly striking was the parallelism found between 86Rb+ leakage and inhibition of protein synthesis by treatment with different genetic variants of thionins (α1 purothionin, α2 purothionin, β purothionin from wheat; hordothionin from barley), as well as with the viscotoxins, which are homologous polypeptides from the European mistletoe.