bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of the Dansylation Reaction of Amino Acids, Peptides and Proteins
Authors: Gros C. , Labouesse B.
Published: July 2010

The reaction rates between dansyl chloride and water, amino acids or peptides are studied as a function of pH and temperature. The rate of hydrolysis of dansyl chloride is constant and low up to pH 9.5 and above this pH it increases rapidly. The various reactive groups of amino acids and peptides react with dansyl chloride in their unprotonated form. It is shown that a compromise for optima conditions of dansylation (pH and temperature) may be found, taking into account the rate of hydrolysis and the pK of the group to be dansylated.

A complete chromatographic separation on Silica gel G thin layer plates of dansyl amino acids is proposed using 4 solvents. The quantitative recovery of the separated derivatives is described.

The study of the acid hydrolysis of dansylated proteins shows that the release of the dansyl derivatives from the peptide chain is faster than that of free amino acids; it also shows that the destruction of dansyl amino acids occurs rather rapidly. Therefore a hydrolysis time of 4 hours (110°) is suggested, except in the special cases of the release of dansyl valine, dansyl leucine or dansyl isoleucine which needs 18 hours of hydrolysis.

For quantitative estimation of each dansyl derivative a correction factor is given, taking into account the loss during hydrolysis, the recovery from the plates and the individual fluorescence of any dansyl derivative as compared to the fluorescence of a reference compound.

The general conditions described in this work require 1 nmole of material for qualitative determination of the N‐terminal amino acids, and 5–10 nmoles for their quantitative estimation.

Some examples (α‐chymotrypsin, trypsinogen, ribonuclease, lysozyme, aspartokinase and triose phosphate dehydrogenase) illustrate the efficiency of the method.
Studies in Phytosterol Biosynthesis: Observations on the Biosynthesis of Fucosterol in the Marine Brown Alga Fucus spiralis
Authors: Goad L. J. , Goodwin T. W.
Published: July 2010

Fucosterol biosynthesised by Fucus spiralis from [2‐14C,(4R)‐4‐3H1] mevalonic acid has a 3H/14C atomic ratio of 3/5. Isomerisation of the labelled fucosteryl acetate gave 3β‐acetoxystigmasta‐5,24‐diene with a 3H/14C atomic ratio of 2/5. This demonstrated that the fucosterol had a 3H atom located at C‐25 and therefore proves that hydrogen (tritium) migration from C‐24 to C‐25 occurs during alkylation of the phytosterol side chain. Cycloartenol and 24‐methylene cycloartanol also isolated from the Fucus spiralis were shown to be radioactively labelled, there was no evidence of radioactive lanosterol. Tritium was shown to be present at C‐3 of the biosynthesised cycloartenol but was absent from C‐3 of the fucosterol.
Studies on the Structures and Apoprotein Linkages of the Phycobilins
Authors: Rüdiger W. , Carra P. Ó
Published: July 2010

The structures of the phycobilins have been studied by chromic acid oxidation followed by identification of the imide products by thin layer chromatography. The results, in conjunction with spectral and other data, permit the assignment to phycoerythrobilin of a structure which may be regarded as a derivative of mesobilidiene a, b IXα, with the ethyl side‐chain and ring double‐bond of ring A replaced by an isomeric ethylidene grouping, and the ethyl side‐chain of ring D replaced by a vinyl group. Phycocyanobilin has an isomeric bilitriene structure which may be regarded as mesobilitriene IXα with the ethyl side‐chain and ring double‐bond of ring A replaced by an ethylidene grouping.

Oxidation of intact algal biliproteins reveals that the covalent linkages of the above two phycobilins to their apoproteins involve both a group in ring A, probably the ring nitrogen group, and a carboxyl group of one of the propionic acid side‐chains.
Déficience en cytochrome oxydase chez les souches de Saccharomyces cerevisiae exigeant la thréonine pour leur croissance
Authors: Surdin Y. , de Robichon‐Szulmajster H. , Lachowicz T. M. , Slonimski P. P.
Published: July 2010

During the study of the biosynthesis of threonine and methionine in Saccharomyces cerevisiae it appeared that the strains carrying the unliked mutated alleles thr2 or thr3 were devoid of cytochrome oxidase activity. Genetic studies have shown that: (a) these strains are not petites; (b) the cytochrome oxidase deficiency segregates with the auxotrophic character for threonine and methionine; (c) reversion to prototrophy simultaneously leads to the recovery of cytochrome oxidase synthesis; (d) the presence of a suppressor for one heteroallele of the thr2 locus leads also to the recovery of cytochrome oxidase synthesis.

Additional studies have shown that partial cytochrome oxidase deficiency is found in mutants blocked at other steps in the threonine biosynthetic pathway. The multiplicity of unlinked genes giving such an effect has led to the hypothesis of a metabolic relationship between the two phenotypes.
N‐Terminal Fragment of Hog Pepsin
Authors: Trufanov V. A. , Kostka V. , Keil B. , Šorm F.
Published: July 2010

One of the peptide fragments obtained in a preceding study of the cyanogen bromide hydrolysate of S‐sulfo‐pepsin has been investigated in more detail. The peptide was purified by ion‐exchange chromatography and obtained in homogeneous form. It contains 158 amino acid residues, among their number the only histidine residue of pepsin and two half‐cystine residues. Evidence was obtained that the peptide is derived from the N‐terminal region of the molecule of hog pepsin. The peptide was reduced by 2‐mercaptoethanol and converted into its S‐(β ‐amino‐ethyl)‐cysteinyl derivative. From the tryptic digest of this derivative a 105‐residue peptide containing N‐terminal isoleucine was isolated which represents the N‐terminal segment of the pepsin molecule.
Insect Immunity. Purification and Properties of Three Inducible Bactericidal Proteins from Hemolymph of Immunized Pupae of Hyalophora cecropia
Authors: HULTMARK Dan , STEINER Håkan , RASMUSON Torgny , BOMAN Hans G.
Published: July 2010

Three inducible bacteriolytic proteins, designated P7, P9A and P9B, from the hemolymph of immunized pupae of the giant silk moth Hyalophora cecropia have been purified using a two‐step procedure with cation‐exchange chromatography. Purified protein P7 has a molecular weight of 15000 and its amino acid composition shows a great similarity to that of the lysozyme from the wax moth Galleria mellonella. Moreover, heat stability, pH‐rate profile and bacteriolytic specificity also indicate that protein P7 is a lysozyme. The other purified proteins, P9A and P9B, are highly potent against Escherichia coli and some other gram‐negative bacteria. The amino acid compositions of proteins P9A and P9B are very similar, although the contents of glutamic acid and methionine were different. The molecular weights of these very basic proteins are around 7000. The P9 proteins are heat stable; their activities were retained after 30 min incubation at 100°C. Both forms of protein P9 clearly differ from the lysozyme class of enzymes and they may represent a new type of bacteriolytic protein.
Synthesis of δ‐Aminolaevulinate Synthase in vitro Using Hepatic mRNA from Chick Embryos with Induced Porphyria
Authors: BROOKER John D. , MAY Brian K. , ELLIOTT William H.
Published: July 2010

Polyadenylated mRNA was isolated from chick embryo liver following induction of hepatic porphyria. The RNA was translated in vitro using a wheat germ cell‐free system and δ‐aminolaevulinate synthase was identified in the translation products by indirect immunoprecipitation. The enzyme was not apparent in the translation products of polyadenylated RNA from non‐induced livers.

The molecular weight of δ‐aminolaevulinate synthase synthesised in vitro was 70000 and the protein was estimated to represent up to 5% of total products synthesised in vitro.

These data demonstrate for the first time that induction of chick embryo liver δ‐aminolaevulinate synthase activity in hepatic porphyria correlates with a large increase in the translational capacity of isolated polyadenylated RNA for this enzyme and, together with preliminary cDNA · RNA hybridization studies, indicate that an increase in the level of δ‐aminolaevulinate synthase mRNA is responsible.
Photoinduced Protein‐RNA Cross‐Linking in Mammalian 80‐S Ribosomes
Authors: REBOUD Anne‐Marie , BUISSON Monique , DUBOST Simone , REBOUD Jean‐Paul
Published: July 2010

RNA‐protein interactions in the 80‐S rat liver ribosomes were studied by measuring cross‐linking of proteins to rRNAs induced by ultraviolet radiation, as already reported for free 40‐S and 60‐S subunits. Our results are compatible with the model in which most of the ribosomal proteins are accessible to rRNAs in the native conformational state of the ribosomes. Subunit association in 80‐S ribosomes does not seem to induce modifications in protein‐RNA interactions as measured by this irradiation technique. However, two proteins, S9 and S13, appeared to be significantly less cross‐linked with RNA in ribosomes than in free subunits.

Ribosomes which had been frozen and thawed several times were highly sensitive to ultraviolet radiation. Such treatment in the cold chiefly modified their 60‐S subunit moiety.
Subunit Composition and Partial Reactions of the 2‐Oxoglutarate Dehydrogenase Complex of Acetobacter xylinum
Authors: De Kok Arie , KORNFELD Saul , BENZIMAN Moshe , MILNER Yoram
Published: July 2010

The 2‐oxoglutarate dehydrogenase complex from Acetobacter xylinum consists of three components, 2‐oxoglutarate dehydrogenase (E1), lipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3). The molecular weight of the complex as studied by sedimentation and diffusion is 2.6 × 106. The E1 component and the E2E3 subcomplex were purified by ultracentrifugation and gel filtration after dissociation of the complex at pH 9.8. The E2E3 subcomplex was further dissociated in 1 M NaCl at neutral pH and the components purified by gel filtration.

The active E1 component is a tetramer of Mr 360000. The E3 component is a dimer of Mr 103000. The E2E3 subcomplex showed an S20,w value of 36.6 S indicating an Mr around 1.6 × 106. The Mr of E2 was estimated to be 900000. From these data and the chain weights as derived from sodium dodecylsulphate gel electrophoresis it was concluded that the complex consists of 12 E1, 12 E2 and 12 E3 chains.

Incorporation of N‐ethyl[2,3‐14C]maleimide in the presence of NADH indicated the presence of 12 reactive SH groups per complex located on the E2 component. Titration of the lipoyl moiety with substoichiometric amounts of N‐ethylmaleimide in the presence of NADH, as assayed by cleavage with 5,5′‐dithio‐bis(2‐nitrobenzoic acid), indicated that the complex contains 12 lipoyl groups of which only one of the two SH groups is reactive towards N‐ethylmaleimide.

In agreement with this number, reaction of the complex with 2‐oxo[2‐14C]glutarate yields incorporation of 12–14 mol succinyl groups/mol complex. With non‐saturating concentrations of 2‐oxo‐glutarate, the presence of the allosteric effector AMP or addition of N‐ethylmaleimide was required for maximum incorporation. AMP caused enhancement of the rate of succinylation with cooperative dependence on the AMP concentration, Hill coefficient h= 1.3 at pH 7.4 and h= 2 at pH 8.2. N‐Ethylmaleimide prevented desuccinylation. The rate of succinylation was at least six times slower than the rate of FAD reduction as measured from the decrease in fluorescence in the presence of coenzyme A. This indicates that either coenzyme A participates in the succinylation of the lipoyl moiety or that the formation of succinyllipoate is a side reaction of the catalytic pathway, which takes only place in the absence of coenzyme A.
Properties of the Interaction of the Sodium Channel with Permeant Monovalent Cations
Authors: JACQUES Yannick , ROMEY Georges , FOSSET Michel , LAZDUNSKI Michel
Published: July 2010

The use of sea anemone toxin, veratridine and scorpion toxin which specifically interact with the gating system of the sodium channel and maintain the channel in an open conformation has permitted a study of the mechanism of transport of monovalent cations through the selectivity filter of this channel. The initial rate of 22Na+ influx through the tetrodotoxin‐sensitive Na+ channels of excitable cells is dependent upon the external concentrations of Na+ and Na+‐substitutes with the following properties. (a) It is saturable at high Na+ concentrations and increases with the external Na+ concentration in a cooperative manner (nH= 1.6). (b) At low external Na+ concentrations (1 mM), it is activated and then inhibited by increasing external concentrations of monovalent cations such as Li+, guanidinium, hydrazinium, hydroxylamine and K+. The activating effect of these cations disappears at higher external Na+ concentrations (10 mM).

The experimental data are consistent with a model involving at least two allosteric cation‐binding sites per Na+ channel. The binding of monovalent cations to Na+ sites is characterized by a high positive homotropic cooperativity.

Most of the work describes the properties of the Na+ channel in neuroblastoma cells. The mechanism has also been shown to be valid for excitable cells of other types and origins.
Stereochemistry of the Conversions of l‐Threonine and d‐Threonine into 2‐Oxobutanoate by the l‐Threonine and d‐Threonine Dehydratases of Serratia marcescens
Authors: CROUT David H. G. , GREGORIO Maria V. M. , MÜLLER Urs S. , KOMATSUBARA Saburo , KISUMI Masahiko , CHIBATA Ichiro
Published: July 2010

dl[3‐2H]Threonine, l‐[3‐2H]threonine, (2RS,3S)‐2‐amino[3‐2H1] butanoic acid, (2RS,3S) 2‐amino[3‐3H1]butanoic acid and dl‐2‐amino[3‐14C]butanoic acid were synthesised.

l‐[3‐2‐H]Threonine was converted into l‐[4‐2H1]isoleucine by Serratia marcescens strain IHr313.

(2RS,3S)‐2‐Amino[3‐2‐H1]butanoic acid was converted into l‐[4‐4‐H1]isoleucine by S. marcescens strain 149.

Analysis by 220‐MHz NMR spectroscopy of the labelled l‐isoleucine produced showed that the same diastereotopic hydrogen at C‐4 was labelled in each experiment, proving that, during the conversion of l‐threonine into 2‐oxobutanoate mediated by biosynthetic l‐threonine dehydratase, the hydroxyl group at C‐3 was replaced by hydrogen with retention of configuration.

S. marcescens strain 149 lacks l‐threonine dehydratase but possesses an inducible d‐threonine dehydratase. This strain converted dl‐[3‐2H]threonine into dl‐[4‐2H1]isoleucine with the deuterium located in the diastereotopic C‐4 hydrogen derived from the 3 pro‐R hydrogen of 2‐aminobutanoic acid.

This result proved that during the conversion of d‐threonine into 2‐oxobutanoate mediated by d‐threonine dehydratase, the hydroxyl group at C‐3 is replaced by hydrogen with retention of configuration.

These results also prove that the protonation at C‐3 of the proposed enamine intermediate in the transformations catalysed by threonine dehydratase is under enzymatic control.

The present results, taken in conjunction with the independent assignment of the signals in the 220‐MHz NMR spectrum of l‐isoleucine due to the diastereotopic protons at C‐4, prove that during the ethyl migration step in l‐isoleucine biosynthesis, the configuration at the migrating centre is retained.
Dark Isomerization of Retinals in the Presence of Phosphatidylethanolamine
Authors: GROENENDIJK Gerard W. T. , JACOBS Cor W. M. , BONTING Sjoerd L. , DAEMEN Frans J. M.
Published: July 2010

Dark incubation of retinoids (retinyl ester, retinol, retinal, retinaloxime) in suspensions of rod outer segment membranes leads to substantial isomerization (and partial degradation) in the case of retinals only.

All‐trans, 13‐cis and 9‐cis‐retinal all isomerize at the Δ13 double bond leading to an equilibrium with approximately 75%trans and 25%cis isomer at this bond (all‐trans⇔ 13‐cis and 9‐cis⇔ 9,13‐dicis).11‐cis‐Retinal isomerizes irreversibly to a mixture of all‐trans and 13‐cis‐retinal.

The active compound appears to be phosphatidylethanolamine present in the membrane. The amino group and the phosphate, as well as the hydrophobic part of the phospholipid are essential.

At least three factors are important for the phosphatidylethanolamine‐catalyzed isomerization as studied with the 13‐cis isomer: the concentration of phosphatidylethanolamine, the concentration of Schiff base between retinal and phosphatidylethanolamine and the presence of lipid aggregates.

Based on these observations a mechanism is proposed, which satisfactorily explains the specificity of the isomerization pattern.

It is suggested that reisomerization of all‐trans to 11‐cis retinal in vivo takes place by fixation of all‐trans retinal on an adequate surface (e.g. opsin) and a localized nucleophilic attack on the C‐11 atom, followed by trapping of the isomerized chromophore by opsin.

It is further concluded that retinal does not occur in vivo as a free intermediate. Direct transfer from one protein to another (opsin, retinol dehydrogenase, retinal binding proteins) seems to take place.
Nucleotide Sequence of the Simian Virus 40 HindII + III Restriction Fragment A (Second Part of the T Antigen Gene)
Authors: VOLCKAERT Guido , VAN DE VOORDE André , FIERS Walter
Published: July 2010

The nucleotide sequence of the second part of the simian virus 40 DNA HindII + III restriction fragment A is presented. The sequence extends from map position 0.533 to 0.424 and together with the first part of Hind‐A [Volckaert et al. Proc. Natl Acad. Sci. U.S.A. 75, 2160–2164 (1978)] completes the total Hind‐A sequence, comprising 1169 base pairs. The second half of Hind‐A includes the region corresponding to the second splicing boundary common to small tumor antigen (small‐t) and large tumor antigen (large‐T) mRNA and it contains coding information for an internal portion of large‐T antigen. Two similar secondary structures of reasonable thermodynamic stability can be proposed for the nucleotide sequence of the pre‐mRNA corresponding to the region reported here. Their possible relevance to the splicing of the SV40 early mRNAs is discussed.

The deduced amino acid sequence is 188 residues long and contains a Lys‐Lys‐Lys‐Arg‐Lys stretch which may be involved in the DNA binding capacity of large‐T. A presumptive phosphorylation site is also present.
Nucleotide Sequence of the Simian Virus 40 HindII + III Restriction Fragment I (Fourth Part of the T Antigen Gene)
Authors: VAN HERREWEGHE Jacqueline , VAN DE VOORDE André , FIERS Walter
Published: July 2010

The HindII + III restriction fragment I (Hind‐I) from simian virus 40 DNA represents 4.96% of the genome and maps in the early transcription region. Hind‐I is an internal segment of the A gene and its information is expressed as part of the early 19‐S mRNA, which codes for T antigen. We report here the nucleotide sequence of the 259‐base‐pair Hind‐I fragment. The sequence was determined and confirmed by RNA and DNA sequencing methods: by analysis of oligonucleotides resulting from T1 and pancreatic RNase digestion of labeled RNA transcribed from SV40 DNA with Escherichia coli DNA‐dependent RNA polymerase, by partial degradation of RNA transcripts with snake venom phosphodiesterase, and by base‐specific chemical degradation of 5′‐end‐labeled subfragments of Hind‐I according to the procedure of Maxam and Gilbert.

Multiple triplets corresponding to termination codons occur in two of the three reading frames of the DNA strand that has the same polarity as early mRNA. The open reading frame connects in phase with the one of the Hind fragments flanking Hind‐I, and the amino acid sequence specified by Hind‐I lies in the middle part of the large‐T antigen. Some features of the primary nucleotide sequence and of early transcription are discussed.
Nucleotide Sequence of the Hind‐I‐proximal Part of Simian Virus 40 HindII + III Restriction Fragment B. Fifth Part of the T Antigen Gene
Authors: VAN HERREWEGHE Jacqueline , VAN DE VOORDE André , FIERS Walter
Published: July 2010

The nucleotide sequence of the simian virus 40 DNA segment that lies between the HindIII restriction endonuclease cleavage site at map position 0.324 and the AtuI cleavage site at 0.261 has been determined by the base‐specific partial chemical degradation procedure of Maxam and Gilbert. This region comprises 335 base pairs and represents 6.4% of the total SV40 genome and 41% of the restriction fragment Hind‐B. It connects in the clockwise direction to the restriction fragment Hind‐I (described in the preceding paper).

Hind‐B is situated in the second half of the early transcription region of the SV40 genome and encodes information (including the termination signal) for the early protein large‐T antigen. In the part of Hind‐B described here, the DNA strand that has the same polarity as early 19‐S mRNA defines only one open reading frame for translation; the other two are blocked by multiple triplets corresponding to termination codons. The open reading frame is part of one that runs throughout much of the early region: from the second splicing boundary of the large‐T gene (position 0.534) to the information for the large‐T stop signal (position 0.174) near the Hind‐B/Hind‐G junction, and which together with the non‐contiguous DNA segment from position 0.65 (site of the information for the large‐T start signal) to 0.60 (the first splicing boundary) codes for the entire large‐T antigen.
Nucleotide Sequence of Simian Virus 40 DNA: Structure of the Middle Segment of the HindII + III Restriction Fragment B (Sixth Part of the T Antigen Gene and Codon Usage)
Authors: VAN HEUVERSWYN Hugo , VAN DE VOORDE André , VAN HERREWEGHE Jacqueline , VOLCKAERT Guido , DE WINNE Paul , FIERS Walter
Published: July 2010

We report here the nucleotide sequence of the simian virus 40 DNA region that lies between the EcoRII restriction endonuclease cleavage sites at map positions 0.214 and 0.281. The sequence was determined by partial chemical degradation of terminally labeled DNA fragments according to the procedure of Maxam and Gilbert. This region represents 6.7% of the SV40 genome and is located in the middle of HindII + III restriction fragment B. It is expressed as part of the early 19‐S messenger RNA, which codes for the large‐T antigen protein. Only one open reading frame for translation can be deduced from the message strand of the DNA and this reading frame connects in phase with the one of both neighboring fragments.

This publication is the last in a series of papers about the T‐antigen gene, and several properties of this gene and its product are discussed. The non‐randomness of codon usage is similar to that previously discussed for the late part of the genome. Moreover, it appears that the choice of a third letter can be determined by the nature of the following codon; some codons which start with a pyrimidine are almost never preceded by an adenosine and some ANN‐type codons are almost never preceded by a guanosine.
The Unmasking of Proteolytic Activity during the Early Development of Artemia salina
Authors: GARESSE Rafael , PERONA Rosario , MARCO Roberto , VALLEJO Carmen G.
Published: July 2010

The proteolytic activites of Artemia salina immediately after hatching are found masked in a precursor of high molecular weight (∼ 100000). The molecular weight of this precursor decreases slightly as development proceeds. However, its kinetic and chromatographic properties vary greatly. Shortly after hatching, the activities are highly inhibited, can be activated severalfold by the chaotropic agent potassium iodide and are not proportional to the amount of enzyme‐containing extract added. Later after hatching, its kinetics become normal. From these observations we have concluded that (a) the majority, if not all, of the proteinase activities found later in development are already present immediately after hatching, although in an inhibited state that is gradually activated; (b) the proteinases are not found free in the cytoplasm but in a complex which may allow regulation of their activity.
Variable Rate of Polypeptide Chain Elongation in vitro
Authors: ABRAHAM Abraham K. , PIHL Alexander
Published: July 2010

Translation of rabbit globin mRNA and tobacco mosaic virus mRNA was studied in a wheat germ system. After a short pulse with labeled methionine, the reaction products were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and visualized by fluorography. Discrete bands corresponding to incomplete products were detected both with globin and tobacco mosaic virus RNA as messenger at a time when no release of peptides from tRNA could be detected.

In the absence of spermidine certain intermediate products accumulated during the chase period.

With increasing incubation time, the radioactivity of most bands was chased into higher‐molecular‐weight products. This change in size distribution was more pronounced when magnesium was partially replaced by spermidine.

Evidence is presented that some of the incomplete products had in part been released from tRNA. No such release of polypeptides could be detected in experiments using the artificial messenger poly(U), indicating that non‐specific peptidyl‐tRNA hydrolases were not present in the system.

The transient accumulation of discrete bands during the chase period indicates that in the wheat germ system polypeptide chain elongation occurs at discontinuous rates. The fact that spermidine increases the rate of elongation and the yield of full‐length translation products may be due to its ability to facilitate the movement of the ribosomes beyond regions where the elongation is retarded.
A New Semi‐empirical Method for the Determination of the Subunit Molecular Weight of a Protein
Authors: KUBOTA Ichiro , TSUGITA Akira
Published: July 2010

We have developed a semi‐empirical method to determine the subunit molecular weight of a protein. The method is a minor modification of the Edman degradation and is based on a simple chemical procedure to modify specifically the N‐terminal NH2 group. Initially, all NH2 groups, including the ɛ‐NH2 groups of lysine residues and the N‐terminal α‐NH2 group, are reacted with phenylisothiocyanate and the protein derivative is subjected to one step of the Edman degradation. The newly exposed N terminus is then reacted with radioactively labelled phenylisothiocyanate. A value for the subunit molecular weight can be obtained from an analysis of the incorporated radioactivity and the amount of the protein in the sample. The molecular weight of five different proteins have been determined by this method. Our method is particularly useful for proteins containing lipid or sugar components and also for relatively small peptides. The procedure described in this paper for the specific modification of the N terminus has been found to be a powerful tool for protein sequencing.
The Primary Structure of the Phosphatidylcholine‐Exchange Protein from Bovine Liver
Authors: MOONEN Peter , AKEROYD Robert , WESTERMAN Jan , PUIJK Wouter C. , SMITS Petrus , WIRTZ Karel W. A.
Published: July 2010

The phosphatidylcholine exchange protein from bovine liver consists of a single polypeptide chain and has a blocked N terminus. The protein contains an estimated 244 amino acid residues in accordance with a determined molecular weight of 28000. The protease from mouse submaxillaris gland cleaved the citraconylated and S‐carboxymethylated derivative of the exchange protein at one specific site (Arg14‐Glu15) close to the N terminus. Analysis of the two resulting peptides showed that N‐acetyl‐methionine was the N‐terminal residue and gave the sequence of the first 41 residues.

The modified protein was also fragmented with the protease from Staphylococcus aureus. The peptides isolated represented 88% of the protein; their sequences were determined by manual and automated Edman degradation. Alignment of a number of these peptides gave the complete sequence of the N‐terminal half up to position 122.
Reactivity of the Thiol Group in Human and Bovine Albumin at pH 3–9, as Measured by Exchange with 2,2′‐Dithiodipyridine
Authors: PEDERSEN Anders Overgaard , JACOBSEN Jørgen
Published: July 2010

The kinetics for exchange between an aromatic disulphide and the thiol group in human and bovine albumin as well as in glutathione were investigated in the pH range 2.5–9.8.

For both albumins the rate constants exhibit a maximum near pH 3, confirming the results of Svenson and Carlsson's investigation of bovine albumin [A. Svenson and J. Carlsson (1975) Biochim. Biophys Acta, 400, 433–438]. This was related to the well known N–F conformational change of the protein.

At pH 5–8 the reactivity of the thiol group in both albumins and glutathione changes sharply, probably due to ionization of the thiol group. At pH above 8, however, the reactivity of the thiol group in albumins, but not in glutathione, becomes nearly independent of pH.

In addition, a conformational change at pH 6.5–8.5 was studied by means of differential spectroscopy of bilirubin, liganded to human albumin. This neutral transition appeared to proceed identically in mercaptalbumin and nonmercaptalbumin.

It is concluded that (a) the pK of the thiol group in albumin is significantly below that of SH in glutathione, and (b) ionization of this thiol group, Cys‐34, is independent of the neutral transition.
Structural Comparisons of Superoxide Dismutases
Authors: HARRIS J. Ieuan , AUFFRET Anthony D. , NORTHROP Frederick D. , WALKER John E.
Published: July 2010

The amino‐terminal sequences of superoxide dismutase isolated from seven microorganisms have been determined. These include the first sequences of enzyme from anaerobic phototrophes. Five enzymes contain iron and two manganese. The enzymes are all related to each other but not to the Cu/Zn family of superoxide dismutases. These sequences, taken with six others from the same family, show that there is no clear distinction in sequence between Fe and Mn types. Moreover it demonstrates a wide variation between enzymes from different bacteria. Also enzymes from anaerobes do not seem to be a particularly closely related group and are not more closely related to each other than to enzymes from aerobes. Two histidine residues are conserved in all proteins and secondary structure predictions suggest they are in close proximity in the same α‐helix. These residues may provide ligands for the bound metal.
Photoreactions of Cytochromes in Algal Chloroplasts
Authors: BÖHME Herbert , KUNERT Karl‐Josef
Published: July 2010

Light‐induced cytochrome redox reactions were investigated with algal chloroplasts capable of high rates of electron transport coupled to phosphorylation. The electron‐donor pool preceding photosystem I consists of membrane‐bound cytochrome f‐554.5 and a soluble cytochrome c‐553; the latter replaces the function of plastocyanin of higher‐plant chloroplasts. Both cytochromes are reduced by photosystem II and oxidized by photosystem I. A site of energy conservation precedes these c‐type cytochromes. The data obtained with respect to the function of b‐type cytochromes are comparable to those obtained with higher‐plant chloroplasts. Cyclic electron transport is mediated by cytochrome b‐563 in a photosystem‐I‐dependent reaction. In addition, cytochrome b‐563 may be reduced by photosystem II, in accordance with recent findings with intact spinach chloroplasts. It therefore appears that cytochrome b‐563 is a member of both cyclic and non‐cyclic electron transport. In contrast to higher‐plant chloroplasts, redox reactions of cytochrome b‐559 are observable without any pretreatments. Cytochrome b‐559, high‐potential, is reduced by photosystem II through plastoquinone. In the presence of carbonyl cyanide m‐chlorophenylhydrazone a cytochrome b‐559 oxidation by photosystem II is measured.
Regulatory Effects of Iodide and Thiocyanate on Tyrosine Oxidation Catalyzed by Thyroid Peroxidase
Authors: MICHOT Jean‐Luc , OSTY Jeannine , NUNEZ Jacques
Published: July 2010

The effects of iodide, thiocyanate and perchlorate, three anions with the same molecular size, on the oxidation of tyrosine to 3,3′‐bityrosine by several peroxidases were evaluated at pH 8.8, i.e. in conditions in which iodide is not oxidized. The following results were obtained:

Iodide greatly stimulates the rate of bityrosine formation in the presence of thyroid peroxidase. No effect was seen with horseradish peroxidase or lactoperoxidase. Maximal iodide effects were obtained with about 0.5 mM iodide and Km for iodide was equal to about 0.028 mM. These results suggest that thyroid peroxidase contains a simple class of regulatory binding sites for iodide.

SCN− mimics iodide effects; maximal stimulatory effects were seen with about 0.5 mM thiocyanate and Km for SCN− was equal to 0.1 mM. The effects of SCN− and those of iodide were not additive. These results suggest that SCN− binds to the same regulatory site as iodide but with a slightly lower affinity. No effect of SCN− was seen with horseradish peroxidase or lactoperoxidase.

ClO−4, another anion with the same molecular size as iodide and SCN−, had neither an effect on the oxidation of tyrosine to bityrosine nor did it prevent the stimulatory effect of iodide on this reaction. Bromide was without effect on the same reaction.
Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii
Authors: VOORDOUW Gerrit , VAN DER VIES Saskia , SCHOLTEN Jan Willem , VEEGER Cees
Published: July 2010

Purified transhydrogenase from Azotobacter vinelandii forms very stable polymers at neutral pH. These show little tendency to dissociate upon dilution but can be disoociated by raising the pH.

The stability of transhydrogenase polymers towards dilution can be demonstrated with enzyme that is in part covalently bound to Sepharose. Non‐covalently bound subunits are kept bound upon elution at pH 7.5 and can only be released by raising the pH of the eluting buffer (pH 9.0). The remaining dissociated immobilized polymers can be reconstituted by adding free transhydrogenase at a lower pH (7.5–8.5). Reconstitution of dissociated immobilized A. vinelandii polymers can also be achieved with enzyme from Pseudomonas aeruginosa, which demonstrates the close structural relationship between these two enzymes.

Differential centrifugation of cell suspensions lysed with the osmotic shock method in the presence of EDTA shows transhydrogenase to be present largely as the non‐polymerized octamer (S20,w= 15 S). However, the distribution of the enzyme activity changes markedly when divalent cations (MgCl2, CaCl2) are included in the lysis buffer: up to 65% of the transhydrogenase activity then co‐sediments with membranes and nucleic acids (30 min, 20000 × g). Nuclease treatment leads to disappearance of the rapidly sedimenting transhydrogenase fraction. These results are incompatible with a reversible divalent‐cation‐mediated self‐association of the enzyme in vivo. We conclude, on the contrary, that the transhydrogenase octamer is partly immobilized in vivo by divalent‐cation‐mediated interactions with cell constituents (e.g. nucleic acids) preventing its self‐association.

The reduction of NAD+ by NADPH catalyzed by cell‐free extract transhydrogenase is not influenced by phosphate ions, while the reverse reaction is completely inhibited by 5 mM phosphate. This inhibition is released by adding excess divalent cations (10 mM CaCl2 or MgCl2). Since other metabolites (adenosine, 5′AMP, ADP, ATP) inhibit both activities more or less to the same extent it follows that the antagonistic action of phosphate and divalent cations is of primary importance in regulating the direction of intracellular transhydrogenation in A. vinelandii.

The existence of binding sites for these ions on the transhydrogenase subunit is indicated by studies on the inhibition of cell‐free extract transhydrogenase with the divalent thiol reagent p‐aminophenylarsenoxide. The rate of reaction is slowed down by magnesium and accelerated by phosphate ions.
The Aldolase‐Substrate Intermediates and Their Interaction with Glyceraldehyde‐3‐phosphate Dehydrogenase in a Reconstructed Glycolytic System
Authors: GRAZI Enrico , TROMBETTA Giorgio
Published: July 2010

The relative concentration of the aldolase · fructose‐bisphosphate and of the aldolase · dihydroxyacetone‐phosphate complexes is regulated, in the steady state, by the nature of the accompanying glycolytic enzymes. Particularly in the presence of triose phosphate isomerase, the aldolase · di‐hydroxyactonephosphate complexes are largely prevalent. This situation is very likely to hold in rabbit muscle in vivo. Aldolase and glyceraldehyde‐3‐phosphate dehydrogenase slowly form a complex; however, no evidence has been found for the direct transfer of glyceraldehyde 3‐phosphate between the two enzymes.
Uptake of Iron from Transferrin by Isolated Rat‐Liver Mitochondria Mediated by Phosphate Compounds
Authors: KONOPKA Krystyna , ROMSLO Inge
Published: July 2010

Isolated rat‐liver mitochondria accumulate iron from transferrin at neutral pH by a mechanism which is markedly stimulated by small‐molecular‐weight polyphosphate compounds. The efficiency of the phosphate compounds decreases in the order: pyrophosphate > ATP > GTP > 2,3‐bis‐(phospho)glycerate > phosphate.

The uptake has a very low energy dependence, and it does not depend on the hydrolysis of ATP or the saturation of transferrin, but it increases in parallel to the concentration of iron to reach a saturation level of 800–1200 pmol iron/mg protein.

Following a chase with unlabelled transferrin, (125I)‐labelled transferrin bound to the mitochondria remains constant, whereas the progressive uptake of 59Fe levels off.

During reincubation of iron‐loaded mitochondria up to 30% of the iron is mobilized in the presence of ascorbate and ATP (or apotransferrin).

The results suggest that iron is mobilized from transferrin by the polyphosphate compounds outside the mitochondria and taken up by the mitochondria in a subsequent reaction.
Visible and Ultraviolet Spectral Transitions of Camphor‐Bound Cytochrome P‐450
Authors: LANGE Reinhard , PIERRE Jacqueline , DEBEY Pascale
Published: July 2010

A systematic analysis of the low‐spin to high‐spin transition in the ultraviolet and visible absorption spectrum of camphor‐bound cytochrome P‐450 is presented. The equilibrium depends on the ionization of two groups, the deprotonation of the first one (pK1= 5.4 at ‐17°C) leading to an increased content of high‐spin type of spectrum, that of the second one (pK2 > 6) to more low‐spin type of spectrum. Both protonation processes and their thermodynamic terms are studied under various conditions of salt, solvent and temperature, including subzero temperatures. The basic mechanism of the transition is the same in aqueous and mixed organic solvent, i.e. the protonation of the first group triggers a conformational change of the whole protein. These phenomena are discussed with reference to functional conformational changes during the catalytic cycle of cytochrome P‐450.
Cytochromes c‐556 from Three Genetic Races of Agrobacterium
Authors: VAN BEEUMEN Jozef , VAN DEN BRANDEN Christiane , TEMPST Paul , DE LEY Jozef
Published: July 2010

The soluble cytochromes c‐556 from three strains of Agrobacterium tumefaciens, B6, II Chrys and Apple 185 have been purified to homogeneity. The strains are representative members of the three main genetic races of Agrobacterium. The purity of the final preparations was established by electrophoresis with an without sodium dodecyl sulphate, by analytical isoelectric focusing and ultracentrifugation, and by N‐terminal analysis.

Properties of these cytochromes were compared with those of cytochrome c‐556 from A. tumefaciens, strain B2a, a member of the same genetic race as strain B6. The four cytochromes are monohaem proteins with molecular weights of about 12300 (determined by four different methods). The isoelectric points of those from strains B6 and B2a are identical at pH 5.5, but they differ from the cytochromes of the other genetic races: cytochrome c‐556 from strain Apple 185 is more acidic (pH 5.2) and that from strain II Chrys more basic (pH 6.2). The cytochromes from strains B6 and B2a have very similar but not identical amino acid compositions; both of them differ more from Apple 185 than from II Chrys c‐556.

Comparison of the tryptic, chymotryptic and thermolytic fingerprints of cytochrome c‐556 from strains B2a and II Chrys reveals strong homology between the primary structures of these cytochromes. Therefore and because of the sequence identity of the first eight residues, the cytochromes c‐556 from strains II Chrys, B6 and B2a are most likely C‐terminal haem‐bound, of the same type as the cytochrome c′ from photosynthetic bacteria.
Cellular Localization of the Apurinic/Apyrimidinic Endodeoxyribonucleases in Rat Liver
Authors: THIBODEAU Lise , VERLY Walter G.
Published: July 2010

A method has been developed to purify rat liver nuclei; the isolated nuclei keep both nuclear membranes and retain more than 90% of the cell apurinic/apyrimidinic (AP) endodeoxyribonuclease activity. The nuclear enzyme is located mostly in chromatin non‐histones; there is also an important amount of activity in the nuclear sap and some in the nuclear membranes. The cytoplasmic AP endodeoxyribonuclease activity is shared between mitochondria, cytosol and membranes. Different cell compartments appear to contain different AP endodeoxyribonuclease species: the membrane enzyme is activated by Triton whereas the other enzymes are rather inhibited; the nuclear sap enzyme has a higher molecular weight and a higher thermal resistance than the chromatin enzyme. A hypothesis is formulated according to which: (1) the chromatin enzyme is the only species important for nuclear DNA repair; (2) the species present in the other cell compartments might be precursors of the chromatin AP endodeoxyribonuclease.
Properties of Amidinated Glucagons
Authors: WRIGHT David E. , RODBELL Martin
Published: July 2010

Porcine glucagon has been reacted with a series of alkyl imidates. The ɛ‐amino group and both the α and ɛ‐amino groups were modified and the subsequent glucagon derivatives were purified by ion‐exchange chromatography and characterized. The modified glucagons were compared with native glucagon in their ability to activate hepatic adenylate cyclase and to compete with 125I‐glucagon for binding to sites specific for glucagon in hepatic plasma membranes. Nɛ‐acetamidino glucagon was as biologically potent, in both activity and binding, as native glucagon, whereas Nɛ‐4 hydroxyphenylamidinoglucagon required a twofold higher concentration to obtain similar levels. These findings suggest that modification through the ɛ‐amino group with alkyl imidates possessing reporter groups should result in glucagon derivatives with significant biological potency, thus providing a new approach to the study of this peptide hormone. Amidination of both ɛ and α‐amino groups resulted in glucagon derivatives which were partial agonists with respect to adenylate cyclase activation and which displayed unexpected anomylous behavior on chromatography.
Physicochemical Properties of the Long‐Chain‐Acyl‐CoA Hydrolase from Rat Liver Microsomes
Authors: BERGE Rolf Kristian
Published: July 2010

The physicochemical properties of a long‐chain‐acyl‐CoA hydrolase (EC 3.1.2.2) of rat liver microsomes have been studied. The hydrolase sedimented as a homogenous species with a sedimentation coefficient, S20, W, of 4.1 S and the molecular weight of 59000 was obtained. Amino acid composition of the enzyme was determined. The specific absorption coefficient, A1%280mn was estimated as 9.8 (1 cm cell length) and the circular dichroic data suggested 50–60% alpha‐helical structure. The enzyme contained three sulphydryl groups, and one of these was exposed to the environment. The hydrolase was inhibited by the serine‐directed reagent phenylmethylsulfonyl fluoride, as well as p‐hydroxymercuribenzoate, N‐ethylmaleimide and 5,5′‐dithiobis(2‐nitrobenzoic acid). Reactivation by means of dithiothreitol was never complete with phenylmethylsulfonyl fluoride and p‐hydroxymercuribenzoate as inhibitors.
Functional Desensitisation of β‐Adrenergic Receptors of Avian Erythrocytes by Catecholamines and Adenosine 3′,5′‐Phosphate
Authors: SIMPSON Ian Alexander , PFEUFFER Thomas
Published: July 2010

Prolonged exposure to β‐adrenergic agonists of pigeon erythrocytes causes a reversible loss (70%) of catecholamine‐stimulated adenylate cyclase activity without reduction in the number of β‐adrenergic receptors. In addition a less pronounced decrease in non‐stimulated and NaF‐stimulated adenylate cyclase activity (15–22%) is observed, appearing at different agonist concentrations and at a different rate. Dibutyryladenosine 3′,5′‐phosphate and the phosphodiesterase inhibitor methylisobutylxanthine partially mimick the action of the β ‐adrenergic agonist, thus pointing to a possible role of adenosine 3′,5′‐phosphate in establishing desensitization. When adenylate cyclase from desensitized cells is stimulated with 5′‐guanylyl‐imidodiphosphate in the presence or absence of catecholamines the lag period preceding the attainment of maximal activity is extended. Likewise the rate of reversal by GTP or ATP of persistent activation of adenylate cyclase is slowed down. This is therefore interpreted to mean that the loss in hormonal ‐stimulation on treatment of pigeon red blood cells with β‐adrenergic agonists is due to a delayed exchange of GDP against GTP on the regulatory GTP‐binding protein. Furthermore, we conclude that events causing the refractory state in avian erythrocytes should occur at a site distal to the β‐adrenergic receptor.
Organization of α‐Globin Genes and mRNA Translation in Subjects Carrying Haemoglobin Hasharon (α47 Asp → His) from the Ferrara Region (Northern Italy)
Authors: del SENNO Laura , BERNARDI Francesco , MARCHETTI Giovanna , PFRROTTA Carla , CONCONI Francesco , VULLO Calogero , SALSINI Giovanna , CRISTOFORI Giulio , CAPPELLOGZA Gino , BELLINELLO Fausto , BEDENDO Bruna , MERCURIATI Maria
Published: July 2010

In subjects carrying the haemoglobin Hasharon mutation (α47 Asp → His), originally from the delta of the Po river (Northern Italy), the concentration of the α‐globin variant has been evaluated and found to be approximately 32%, a value definitely higher than that reported for the same mutant haemoglobin in other regions. Restriction enzyme analysis has been carried out on the DNA from these subjects; the data obtained indicate the presence of three α‐globin genes per diploid cell. Family studies further show that the two normal genes are located on one chromosome and the Hasharon gene on the other. The origin of the single α‐gene in the Hasharon‐carrying subjects of the Ferrara region is discussed in connection with their haematological and biosynthetic data.
Sialoglycoproteins from the Eggs of Pacific Herring
Authors: INOUE Sadako , IWASAKI Mariko
Published: July 2010

A family of hitherto undescribed sialoglycoproteins has been isolated from the eggs of Pacific herring in a yield about 10 times as much as phosvitin known as a major protein component of the eggs. The sialoglycoproteins are composed of 60–50% of carbohydrate and 40–50% of protein. The carbohydrate composition is similar to that of glycoproteins known to have complex types of heterosaccharide units whereas the amino acid composition is unique. The molecular weights ranging over 8800–13000 were determined for the sialoglycoprotein fractions by sedimentation equilibrium. The low mobility of the sialoglycoproteins during sodium dodecyl sulfate gel electrophoresis is discussed in terms of low values of hydrophobicity.