bluuebunny/crossref_metadata_2025 huggingface dataset:
Species‐Specific Inhibition of Homologous Enzymes by Modification of Nonconserved Amino Acids Residues
Authors: Garza‐Ramos Georgina , PÉrez‐Montfort Ruy , Rojo‐DomÍnguez Arturo , de GÓmez‐Puyou Marietta Tuena , GÓmez‐Puyou Armando
Published: July 2004

The possibility of using non‐conserved amino acid residues to produce selective inhibition of homologous enzymes from different species has been further explored with triosephosphate isomerase. S ‐phenyl‐p ‐toluenethiosulfonate (MePhSO2‐SPh), which produces phenyl disulfides with accessible Cys residues, inhibits the activity of rabbit triosephosphate isomerase. The inhibition is due to derivatization of one of the five Cys residues of rabbit triosephosphate isomerase. The effect of MePhSO2‐SPh on triosephosphate isomerase from Saccharomyces cerevisiae, Escherichia coli, chicken and Schizosaccharomyces pombe was also determined. MePhSO2‐SPh did not affect the activity of triosephosphate isomerase from S. cerevisiae and E. coli but it inhibited triosephosphate isomerase from chicken and S. pombe. From an analysis of the Cys content of the various triosephosphate isomerases, it was evident that amongst the ones studied only those that have a Cys in position 217 (or in an equivalent position) were sensitive to MePhSO2‐SPh. Methyl metanethiosulfonate (MeSO2‐SMe), which produces methyl disulfides, had no effect on triosephosphate isomerases that lack Cys217 (S. cerevisiae and E. coli). In triosephosphate isomerases that have Cys217, MeSO2‐SMe inhibited by 40–50% the activity of that from S. pombe, 20–25% that from rabbit but had no effect on the chicken enzyme. In the three latter triosephosphate isomerases, MeSO2‐SMe protected against the strong inhibiting action of MePhSO2‐SPh. The latter observations suggest that MeSO2‐SMe and MePhSO2‐SPh derivatize the same Cys and that significant inhibition of activity requires perturbation by the relatively large phenyl group. The intrinsic fluorescence of rabbit triosephosphate isomerase that had been derivatized to a phenyl disulfide was almost identical to that of the native enzyme. Thus, modification of Cys217 did not produce gross structural alterations, albeit it brought about important kinetic alterations, i.e. a nearly fivefold increase in the Km for glyceraldehyde 3‐phosphate and a 65% decrease in Vmax The effect of derivatizating Cys217 differs markedly from that produced by derivatization of Cys14 (another non‐conserved cysteine). The differences may be explained from their position in the three‐dimensional structure of the enzyme.
1H‐NMR and 13C‐NMR Investigation of Complexes of Mn2+ with Ocytocin Analogues in (2H6)Dimethylsulfoxide
Authors: Valensin Gianni , Maccotta Antonella , Gaggelli Elena , Grzonka Zbigniew , Kasprzykowski Franciszek , Kozlowski Henryk
Published: July 2004

Several ocytocin analogues were synthesised by substitution of the Pro residue with sarcosine or N‐methylalanine, the glutamine residue with threonine and one of the cysteines with 2‐mercaptopropionic acid. All the derivatives were investigated by NMR in dimethylsulfoxide solutions and evidence was obtained for similar preferred conformations in the solution free state. All peptides were shown to form complexes with Mn2+ in solution by the strong paramagnetic effects experienced by several proton resonances. Two structures could be determined, one formed by peptides containing threonine and the other by the remaining peptides. The two structures were delineated by molecular modelling using the Mn2+‐proton distances obtained by NMR as restraints.
Crystal Structure of the Bifunctional Soybean Bowman‐Birk Inhibitor at 0.28‐nm Resolution
Authors: Voss R.‐Holger , Ermler Ulrich , Essen Lars‐Oliver , Wenzl Gabriele , Kim Young‐Mi , Flecker Peter
Published: July 2004

The Bowman‐Birk inhibitor from soybean is a small protein that contains a binary arrangement of trypsin‐reactive and chymotrypsin‐reactive subdomains. In this report, the crystal structure of this anticarcinogenic protein has been determined to 0.28‐nm resolution by molecular replacement from crystals grown at neutral pH. The crystal structure differs from a previously determined NMR structure [Werner, M. H. & Wemmer, D. E. (1992) Biochemistry 31, 999–1010] in the relative orientation of the two enzyme‐insertion loops, in some details of the main chain trace, in the presence of favourable contacts in the trypsin‐insertion loop, and in the orientation of several amino acid side chains. The proximity of Met27 and Gln48 in the X‐ray structure contradicts the solution structure, in which these two side chains point away from each other. The significant effect of a Met27?Ile replacement on the inhibitory activity of the chymotrypsin‐reactive subdomain agrees with the X‐ray structure. Exposed hydrophobic patches, the presence of charged amino acid residues, and the presence of water molecules in the protein interior are in contrast to standard proteins that comprise a hydrophobic core and exposed polar amino acids.
N,N′ Bisformyl Dityrosine is an in vivo Precursor of the Yeast Ascospore Wall
Authors: Briza Peter , Kalchhauser Hermann , Pittenauer Ernst , Allmaier Günter , Breitenbach Michael
Published: July 2004

The amino acid, dityrosine, is a major component of the spore wall surface of the yeast Saccharomyces cerevisiae, where it is part of a highly cross‐linked macromolecular network of yet unknown chemical structure, consisting mostly of glucosamine, dityrosine and few other amino acids. Biosynthesis of the dityrosine moiety of this network consists of several steps, including the chemical modification of free L‐tyrosine and the subsequent oxidative cross‐linking of the modified tyrosine residues (catalyzed by a cytochrome P ‐450), leading to soluble dityrosine‐containing spore wall precursors. We isolated, purified and characterized the dityrosine‐containing precursor that appears late in spore wall synthesis and that is thought to be directly incorporated into the maturing spore wall. Chemical and spectroscopic analyses showed that this precursor is N,N′ ‐bisformyl dityrosine. In addition, we identified a tyrosine‐containing spore wall precursor as N‐formyl tyrosine. The elucidation of the chemical structure of soluble spore wall precursors is crucial for the characterization of the function of the enzymes involved in maturation of the spore surface, e.g. by in vitro systems. A dityrosine‐containing fragment, which was solubilized from mature spore walls by partial hydrolysis, was identified as N‐formyl dityrosine. Mature spore walls contain significant amounts of N‐formyl dityrosine and N,N′ ‐bisformyl dityrosine. This supports the assumption that the dityrosine‐containing macromolecular network on the spore surface has an unusual, non‐peptidic structure.
Characterization of the Type‐1 Repeat from Thyroglobulin, a Cysteine‐Rich Module Found in Proteins from Different Families
Authors: Molina Franck , Bouanani Majida , Pau Bernard , Granier Claude
Published: July 2004

The amino acid sequence of human thyroglobulin is known to enclose cysteine‐rich repetitive regions. In this study, we report the existence of an eleventh type‐1 repeat within the human thyroglobulin sequence, and we characterize the thyroglobulin type‐1 repeat as a protein module. The 11 thyroglobulin type‐1 repeats possessed the same number of cysteine residues (six in type A, four in the two type B repeats), a fairly constant number of residues between cysteines and a conserved sequence pattern. By scanning protein sequence databases, 29 proteins belonging to six different families were found to enclose at least one, and up to three, thyroglobulin type‐1 repeats in their sequence. Although the repeat was present in numerous proteins possessing binding properties, an examination of the information available in the literature showed that a direct role of the repeat in protein‐protein interaction has rarely been assessed. A distance analysis of the sequences indicated that all repeats segregate into four clusters of phylogenically close sequences. A consensus sequence of type‐1 repeats was derived from sequence similarity analysis; it comprised a central core of conserved residues including two highly conserved motifs, QC and CWCV. The type‐1 repeat from thyroglobulin was found to differ from several previously described cysteine‐rich modules, in particular from the epidermal‐growth‐factor‐like module with which it has sometimes been confused. Therefore, our results provide a complete characterization of the repeats which will help in the detection of these repeats in newly characterized proteins, a necessary step for understanding the structural/biological role of this module.
Analysis of the 1H‐NMR Chemical Shifts of Cu(I)‐, Cu(II)‐ and Cd‐Substituted Pea Plastocyanin
Authors: Ubbink Marcellus , Lian Lu Yun , Modi Sandeep , Evans Philip A. , Bendall Derek S.
Published: July 2004

To compare cadmium‐substituted plastocyanin with copper plastocyanin, the 1H‐NMR spectra of CuI‐, CuII‐ and Cd‐plastocyanin from pea have been analyzed. Full assignments of the spectra of CuI‐and Cd‐plastocyanin indicate chemical shift differences up to 1 ppm. The affected protons are located in the four loops that surround the Cu site. The largest differences were found for protons in the hydrogen bond network which stabilizes this part of the protein. This suggests that the chemical shift differences are caused by very small but extensive structural changes in the network upon replacement of CuI by Cd.

For CuII‐plastocyanin the resonances of 72% of the protons observed in the Cul form have been identified. Protons within ?0.9 nm of the CuII were not observed due to fast paramagnetic relaxation. The protons between 0.9–1.7 nm from the CuII showed chemical shift differences up to 0.4 ppm compared to both CuI‐ and Cd‐plastocyanin. These differences can be predicted assuming that they represent pseudocontact shifts. When corrected for the pseudocontact shift contribution, the CuII‐plastocyanin chemical shifts were nearly all identical within error to those of the Cd form, but not of the CuI‐plastocyanin, indicating that the CuII‐plastocyanin structure, in as far as it can be observed, resembles Cd‐ rather than CuI‐plastocyanin. In a single stretch of residues (64–69) chemical shift differences remained between all three forms after correction.

The fact that pseudocontact shifts were observed for protons which were not broadened may be attributable to the weaker distance dependence of the pseudocontact shift effect compared to paramagnetic relaxation. This results in two shells around the Cu atom, an inner paramagnetic shell (0–0.9 nm), in which protons are not observed due to broadening, and an outer paramagnetic shell (0.9–1.7 nm), in which protons can be observed and show pseudocontact shifts.

It is concluded that Cd‐plastocyanin is a suitable redox‐inactive substitute for Cu‐plastocyanin.
Cloning and Sequencing of Human gp330, a Ca2+‐Binding Receptor with Potential Intracellular Signaling Properties
Authors: Hjälm Göran , Murray Edward , Crumley Gregg , Harazim William , Lundgren Stefan , Onyango Isaac , Ek Bo , Larsson Mårten , Juhlin Claes , Hellman Per , Davis Hugh , Åkerström Göran , Rask Lars , Morse Buzzy
Published: July 2004

We present here the complete primary structure of human gp330, the human variant of the principal kidney autoantigen causing Heymann membranous glomerulonephritis in rats. The deduced 4655 amino acid residues give a calculated molecular mass of 519636 Da for the mature protein and consists of a probable 25‐amino‐acid N‐terminal signal peptide sequence, an extracellular region of 4398 amino acids, a single transmembrane‐spanning domain of 23 amino acids, and an intracellular C‐terminal region of 209 amino acid residues. Three types of cysteine‐rich repeats characteristic of the low‐density lipoprotein receptor (LDLR) superfamily are present in human gp330. In the extracellular region, there are a total of 36 LDLR ligand‐binding repeats, comprising four distinct domains, 16 growth factor repeats separated by eight YWTD spacer regions, and one epidermal growth factor‐like repeat. No consensus cleavage sequence for the processing endoprotease furin is detected in human gp330. The intracellular tail contains not only two copies of the F(X)NPXY coated‐pit mediated internalization signal characteristic of LDLR superfamily members, but also intriguing and potentially functional motifs including several Src‐homology 3 recognition motifs, one Src‐homology 2 recognition motif for the p85 regulatory subunit of phosphatidylinositol 3‐kinase, and additional sites for protein kinase C, casein kinase II and CAMP‐/cGMP‐dependent protein kinase. There is approximately 77% amino acid identity between human and rat gp330 with minor differences between the extracellular and intracellular regions. Recently gp330 has been implicated in Ca2+ regulation in the parathyroid, the placenta, and the renal tubule, but its overall physiological and pathological role still remains uncertain.
Changes of Creatine Kinase Secondary Structure Induced by the Release of Nucleotides from Caged Compounds
Authors: Raimbault Cyrille , Buchet René , Vial Christian
Published: July 2004

Light‐induced release of ADP and ATP from their respective caged nucleotides produced small distinct difference infrared spectra of creatine kinase (CK), indicating that ADP and ATP binding to CK promoted different structural alteration. The positive band at 1638–1640 cm−1 and the negative band at about 1650–1652 cm−1 on the reaction‐induced infrared difference spectra in the amide I region were insensitive to the deuteration effects. They were assigned to the peptide backbone of the ADP/ATP‐binding site. In addition Pi or ATP binding produced another positive band at 1657–1659 cm−1 corresponding to the C = O (amide I band) associated with the γ‐phosphate of ATP. This site was also affected when ADP was added, indicating coupling interactions between both sites. No additional structural changes were observed when creatine and ADP were added, suggesting that the creatine‐binding site was uncoupled from the ADP‐binding site. The infrared difference spectra of a transition‐state‐analog complex formed by the addition of ADP, creatine and NO3− (a planar‐phosphate‐mimicking group) lacked the 1657–1659‐cm−1 band indicating that the binding site of γ‐phosphate within CK, was not affected. Infrared changes in the 1560–1590‐cm−1 region suggested that carboxylate groups of Asp or Glu were involved in the binding of Pi, ADP and ATP.
A Site‐Directed Mutagenesis Study of Saccharomyces cerevisiae Pyrophosphatase
Authors: Heikinheimo Pirkko , Pohjanjoki Pekka , Helminen Anne , Tasanen Merja , Cooperman Barry S. , Goldman Adrian , Baykov Alexander , Lahti Reijo
Published: July 2004

We report the expression and initial characterization of 19 active‐site variants of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase), including measurements of thermostability, oligomeric structure and specific activity at pH 7.2. 13 of the 19 conservative substitutions resulted in at least a fivefold decrease in activity, indicating that these residues are important for yeast PPase catalysis. The E58D, D117E, D120E and D152E variants had no activity under the conditions tested, suggesting that Glu58, Asp117, Asp120 and Asp152 may have crucial roles in catalysis. The effects of the mutations on catalytic activity were very similar to those observed with the corresponding variants of Escherichia coli PPase, proving conclusively that the active site and mechanism of soluble PPases are conserved. The D71E variant was more thermostable and the K56R, R78K, D115E and K154R variants were more thermolabile than the wild‐type enzyme, whereas subunit:subunit interactions were somewhat weakened by the K56R, R78K, Y89F and K154R substitutions. These results suggest that Lys56, Asp71, Arg78, Tyr89, Asp115 and Lys154 are structurally important for yeast PPase.
Reversible Inhibition of Sheep Liver Sorbitol Dehydrogenase by Thiol Compounds
Authors: Lindstad Rune I. , Mckinley‐Mckee John S.
Published: July 2004

Reversible inhibition of sheep liver sorbitol dehydrogenase by various thiol compounds has been studied. Most species inhibit the enzyme‐catalyzed reaction competitively with respect to sorbitol, due to the formation of ternary enzyme‐NAD‐thiol complexes. The primary interaction of thiol inhibitors with the enzyme active site involves the catalytic zinc atom, and a bidentate mode of binding to the active‐site metal is indicated for some bifunctional thiols in their ternary complexes. Enzyme‐bound thiolate facilitates NAD binding to the enzyme and vice versa, mainly due to mutual electrostatic stabilization. The aromatic thiols 1‐thio‐1‐phenylmethane and 1‐thio‐2‐phenylethane are especially potent inhibitors with an inhibition constant of 0.30 μM at pH 9.9. The inhibitory effect of aliphatic thiols, which is positively correlated with alkyl chain length, parallels that observed previously with the related enzyme horse liver alcohol dehydrogenase and indicates that interaction with an enzymic hydrophobic site is important for inhibitor binding. Several reversible inhibitors afford competitive protection against affinity labelling of the enzyme by 2‐bromo‐3‐(5‐imidazolyl) propionic acid due to the formation of binary enzyme‐thiol complexes.

The present study establishes thionucleosides as a novel class of potent sorbitol dehydrogenase inhibitors. The thionucleosides 6‐thioguanosine and 6‐thioinosine gave mixed inhibition with respect to sorbitol, due to the formation of enzyme‐NAD‐inhibitor and enzyme‐NADH‐inhibitor complexes.

In order to enable a correlation of the substrate and inhibitor specificities of the enzyme, the kinetic constants for several sorbitol dehydrogenase substrates were determined. l‐threitol and dl‐1–phenyl‐1, 2‐ethanediol are good substrates with, at high pH, kinetic constants similar to those of sorbitol. The potent inhibition by dithiothreitol and the aromatic thiols thus parallels the substrate specificity of the enzyme. The sorbitol competitive inhibitor I‐thiosorbitol is also a substrate with, at pH 7.4, a maximum velocity of 0.17 s‐1 and a Michaelis constant of 8.6 mM.

Dithiothreitol forms a tight ternary complex with the enzyme‐NAD complex with a molar absorbance of 16.4 · 103 M−1· cm‐1 at 311 nm. A spectrophotometric titration of the enzyme with NAD in the presence of dithiothreitol is described, which enables an accurate determination of the concentration of sorbitol dehydrogenase active sites and confirms the activity assay of the enzyme.
The Multiple Forms of Starch‐Branching Enzyme I in Solanum Tuberosum
Authors: Khoshnoodi Jamshid , Blennow Andreas , Ek Bo , Rask Lars , Larsson Håkan
Published: July 2004

Western blot analysis showed the presence of three forms of starch‐branching enzyme (SBE), with apparent molecular masses of 103, 97 and 80 kDa, in extracts of leaves and stored tubers of Solanum tuberosum. The 80‐kDa form was absent in extracts of fresh tuber. Active 80‐kDa enzyme was partially purified from stored tubers and sequence analysis showed that it, similar to the two larger enzyme forms, was an SBE‐I isoform. Limited proteolysis of isolated 103‐kDa SBE‐I under native conditions removed approximately 200 amino acid residues from the carboxy terminus. A stable intermediate with an apparent molecular mass of approximately 80 kDa was formed. Since the 80‐kDa form displayed full enzymatic activity and its circular‐dichroism spectrum did not differ significantly from that of the 103‐kDa enzyme, the carboxy‐terminal portion of the enzyme was suggested to have an extended, unordered structure and therefore to be easily accessible to proteolysis. A cDNA sequence encoding a mature SBE‐I was amplified from tuber mRNA of S. tuberosum by means of PCR. The 3′ end of this sequence differed significantly from that of previously published data. PCR amplification and DNA sequencing of the 3′ ends of the sbeI gene showed that four sbeI alleles exist in the cultivar studied. Two of these four alleles, sbeIa and sbeIb, had slightly longer 3′ ends compared with the other two, sbeIc and sbeId. The difference between the two groups of alleles was due to a partial deletion in sbelc and sbeld of a segment duplicated in all alleles. All four alleles were expressed in leaf and tuber.
The Corrinoid‐Containing 23‐kDa Subunit MtrA of the Energy‐Conserving N5‐Methyltetrahydromethanopterin:coenzyme M Methyltransferase Complex from Methanobacterium Thermoautotrophicum
Authors: Harms Ulrike , Thauer Rudolf K.
Published: July 2004

N
5‐Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum is a membrane‐associated enzyme complex that catalyzes an energy‐conserving, sodium ion translocating step in methanogenesis from H2 and CO2. The complex is composed of eight different subunits, MtrA–H1 one of which (MtrA) harbours a corrinoid as prosthetic group. In this study, we report the structural properties of MtrA1[des‐(214–239)‐MtrA], which is a deletion mutant of MtrA that lacks the last 25 C‐terminal hydrophobic amino acids rendering the membrane protein soluble: (a) mtrAl was heterologously expressed in Escherichia coli. Overexpression yielded a cytoplasmic protein which was purified approximately tenfold to apparent homogeneity. The purified protein was devoid of its corrinoid prosthetic group and not correctly folded as was evident from its electrophoretic mobility in SDS/PAGE. (b) Unfolding of MtrA1 with guanidine/HCl and refolding in the presence of cobalamin resulted in the formation of the correctly folded MtrA1 holoprotein that contained tightly bound cob(II)‐alamin; the rate of reconstitution was highest when the refolding proceeded in the presence of titanium(III) citrate, which suggested that cob(1)alamin is the corrinoid species that binds to the apoprotein. (c) EPR spectra of the cob(II)alamin‐containing holoprotein differentially labelled with 14N (nuclear spin 1) and 15N (nuclear spin 1/2) revealed that the corrinoid is bound to MtrA1 in the base‐off form and that the Co(II) of the prosthetic group is coordinated by a histidine residue of the apoprotein. The results are interpreted with respect to the mechanism of energy conservation by the MtrA–H complex.
The Mechanism of High‐Yielding Chiral Syntheses Catalysed by Wild‐Type and Mutant Forms of Aspartate Aminotransferase
Authors: Contestabile Roberto , John Robert A.
Published: July 2004

The ability of aspartate aminotransferase to catalyse β‐elimination of α‐amino acids that have a good leaving group at Cβ has been exploited in the synthesis of novel amino acids by the inclusion of appropriate nucleophiles as co‐substrates. Two compounds, l‐serine O‐sulphate and 3‐chloro‐l‐alanine, were used as β‐elimination substrates. Nucleophiles used successfully as co‐substrates were thiosulphate, 2‐mercap‐toethanol, mercaptoacetate and aminoethylthiopseudourea. The synthesis achieved using serine O‐sulphate and thiosulphate was found to produce sulphocysteine with a yield of 70%. Circular dichroism demonstrated that the compound was a single enantiomer and, therefore, that nucleophilic addition had taken place on the enzyme. The initial rate of synthesis was 10% of the rate at which the enzyme catalyses its normal transamination reaction. The synthetic reaction was accompanied by minor side reactions that led to small amounts of additional amino acid and oxo acid products through partitions of the main reaction at two stages in the mechanism. By mutating Arg292, which is the residue that binds the distal carboxyl group of natural substrates, the wild‐type enzyme was converted to a form that could discriminate completely between serine O‐sulphate and chloroalanine as β‐eliminating substrate. Similar alterations in nucleophile cosubstrate specificity were also observed. Whereas, for example, the wild‐type enzyme catalysed syntheses between 3‐chloroalanine and either mercaptoethanol or mercaptoacetate with equal facility, the Arg292Asp enzyme showed complete preference for mercaptoethanol. The system should be of general use in the synthesis of novel amino acids as single enantiomers with potentially interesting biological activities.
The Molybdenum Formylmethanofuran Dehydrogenase Operon and the Tungsten Formylmethanofuran Dehydrogenase Operon from Methanobacterium Thermoautotrophicum
Authors: Hochheimer Andreas , Linder Dietmar , Thauer Rudolf K. , Hedderich Reiner
Published: July 2004

Methanobacterium thermoautotrophicum contains a tungsten formylmethanofuran dehydrogenase (FwdABCD) and a molybdenum formylmethanofuran dehydrogenase (FmdABC). The fwdHFGDACB operon encoding the tungsten enzyme has recently been characterized. We report here on the structure and expression of the gene cluster encoding the molybdenum enzyme. This gene cluster is composed of three open reading frames (fmdECB). The fmdB gene was found to encode the molybdopterin‐dinucleotide‐binding subunit harboring the enzyme's active site; FmdB is thus functionally equivalent to FwdB. fmdC encodes a protein with sequence similarity to FwdC in its N‐terminal part and with sequence similarity to FwdD in its C‐terminal part; FmdC is thus functionally equivalent to FwdC and FwdD. Interestingly, the fmd operon lacks a gene fmdA encoding the subunit FmdA of the molybdenum enzyme. FmdA has the same apparent molecular mass and the same N‐terminal amino acid sequence as FwdA and only one DNA sequence encoding for this N‐terminal amino acid sequence was found in the M. thermoautotrophicum genome. It is therefore proposed that FmdA and FwdA are encoded by the same gene namely fwdA in the fwd operon. In agreement with this proposal is the finding that fwdA is expressed constitutively: northern‐blot analysis of RNA from tungstate‐ and molybdate‐grown cells of M. thermoautotrophicum revealed that the fwdHFGDACB gene cluster is transcribed in the presence of either molybdate or tungstate in the growth medium whereas the fmdECB gene cluster was only transcribed when molybdate was present.
Interaction of the Lantibiotic Nisin with Membranes Revealed by Fluorescence Quenching of an Introduced Tryptophan
Authors: Martin Isabelle , Ruysschaerti Jean‐Marie , Sanders Dale , Giffard Catriona J.
Published: July 2004

Nisin is a lantibiotic produced by strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of gram‐positive bacteria. To aid understanding of its mode of action, the interaction of nisin with vesicles of differing phospholipid composition were investigated by fluorescence techniques, using a variant of nisin in which the isoleucine at position 30 was replaced by a tryptophan residue. Activity of the site‐directed variant containing tryptophan was established to be similar to that of the wild‐type peptide. Fluorescence experiments showed a blue shift of the emission wavelength maximum in the presence of lipid vesicles, indicating that the tryptophan residue enters a more hydrophobic environment. Quenching experiments with aqueous and membrane‐restricted quenchers (iodide and spin‐labelled lipids, respectively) both confirmed a non‐aqueous environment for the Trp30 residue, and implied that the residue resides between 0.36 nm and 0.52 nm from the centre of the membrane, depending on the lipid identity. The results clearly demonstrate that nisin interacts strongly with the hydrophobic phase of lipid vesicles. This interaction is stronger in the presence of negatively charged lipids suggesting their importance in the functional interaction of nisin with membranes.
Cloning, Analysis and Inactivation of the ndhK Gene Encoding a Subunit of NADH Quinone Oxidoreductase from Anabaena PCC 7120
Authors: Howitt Crispin A. , Whelan James , Price G. Dean , Day David A.
Published: July 2004

The function of the type‐1 pyridine nucleotide dehydrogenase (NDH‐1) in the cyanobacterium Anabaena PCC 7120 was investigated. Immunological analysis with antibodies raised against NdhK from Synechocystis PCC 6803, a subunit of NDH‐1, showed that NdhK in Anabaena PCC 7120 is only present on the plasma membrane, which confirms the results of previous studies [Howitt, C. A., Smith, G. D. & Day, D. A. (1993) Biochim. Biophys. Acta 1141, 313–320]. Southern analysis with probes from the operon encoding ndhC‐K‐J from Synechocystis PCC 6803 showed that this operon is also conserved in Anabaena PCC 7120. Part of the operon was amplified using PCR with degenerate primers designed against two sequences encoding regions of NdhC and NdhJ that are conserved between cyanobacteria and chloroplasts. The nucleotide sequence of ndhK encodes a protein of 245 amino acids with a predicted molecular mass of 27.5 kDa. The coding regions of ndhC and ndhK overlap by 7 bp, as found in the chloroplasts of liverwort, maize, and rice. This is markedly different from the case in Synechocystis PCC 6803 where a 71‐bp non‐coding, intergenic spacer region lies between ndhC and ndhK. The ndhK clone was interrupted by the insertion of a kanamycin‐resistance gene and used to transform Anabaena PCC 7120. 20 unsegregated transformants were produced, all of which died during attempts to segregate them. This indicates that under the selection conditions used, ndhK is an essential gene in Anabaena PCC 7120.
Role of the Insulin Receptor C‐Terminal Acidic Domain in the Modulation of the Receptor Kinase by Polybasic Effectors
Authors: Baron Véronique , Gual Philippe , Alengrin Françoise , Van Obberghen Einmanuel
Published: July 2004

Basic polymers such as polylysine have been found to activate insulin receptor autophosphorylation and kinase activity toward substrates. It was suggested that acidic receptor domains may be involved in the interaction of the receptor with these basic effectors. In a previous study, we have shown that the receptor acid‐rich C‐terminal sequence, including residues 1270–1280, is involved in the regulation of the receptor kinase activity. Moreover, this domain may be the site of interaction with histone, which is a modulator of the receptor kinase.

In this study, we investigated whether the insulin receptor domain comprising amino acids 1270–1280 is involved in the interaction with polybasic effectors. We used anti‐peptide serum directed to this sequence, and basic activators such as polylysine, polyarginine and protamine sulfate. Our antibodies inhibit polylysine‐induced receptor autophosphorylation, whereas they have no effect on receptor phosphorylation stimulated by concanavalin A which is a non‐basic activator of the insulin receptor. Polylysine‐induced receptor aggregation was blocked by the antibodies (Fab fragments or whole Ig), indicating that competition occurs between the antibody and polylysine at the level of their binding site to the receptor. Finally, we observed a direct interaction of the 125I‐peptide corresponding to receptor sequence 1270–1280 with the basic polymers in dot‐blot experiments. Interestingly, the peptide did not bind spermine, a basic molecule which is not an activator of the insulin receptor kinase.

Our data indicate that the insulin receptor C‐terminal acidic domain including residues 1270–1280 is involved in the interaction of polylysine and other polybasic molecules with the receptor. Since this receptor region has been implicated in the regulation of the receptor kinase activity, we propose that interaction of basic effectors with this domain may be responsible for their activating properties.
Biosynthesis and N‐glycosylation of Human Interferon‐γ
Authors: Sareneva Timo , Mørtz Ejvind , Tölö Hannele , Roepstorff Peter , Julkunen Ilkka
Published: July 2004

Interferon‐γ (IFN‐γ) is a secretory glycoprotein produced by T cells in response to antigenic or mitogenic stimuli. We studied the kinetics of the synthesis, N‐glycosylation, and secretion of IFN‐γ in human CD8+ T lymphocytes stimulated via T‐cell receptor. Highly elevated IFN‐γ mRNA levels were found as early as 1 h after stimulation. Maximal IFN‐γ protein synthesis was observed 2–4 h after induction and appeared to correlate to steady‐state IFN‐γ mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN‐γ from T cells was very rapid, the secretion half‐time being approximately 20–25 min. Inhibition of N‐glycosylation by tunicamycin dramatically reduced the expression of IFN‐γ, but did not block its secretion. Natural IFN‐γ is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell‐free translation/glycosylation system with mutated IFN‐γ constructs lacking either one of the potential glycosylation sites suggested that Asn25 is more efficiently glycosylated than Asn97. Site‐specific oligosaccharide analysis of natural IFN‐γ by glycosidase treatment followed by matrix‐assisted‐laser‐desorption‐ionization mass spectrometry revealed considerable variation in the carbohydrate structures, with more than 30 different forms. The glycans at Asn25 consisted of fucosylated, mainly complex‐type oligosaccharides, whereas the glycans at Asn97 were more heterogeneous, with hybrid and high‐mannose structures. Our results emphasize the essential role of N‐linked glycans in the biology of IFN‐γ and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.
Sequence‐Selective Intercalation of Antitumour Bis‐Naphthalimides into DNA
Authors: Bailly Christian , Braña Miguel , Waring Michael J.
Published: July 2004

LU 79553, a bis‐naphthalimide drug highly active against human solid tumour xenografts, has been shown to bis‐intercalate into DNA with a helix‐unwinding angle of 37°. Footprinting experiments with DNase I reveal that the drug is selective for mixed nucleotide sequences characterised by an alternating purine‐pyrimidine motif, particularly those containing GpT (ApC) and TpG (CpA) steps. Derivatives bearing nitro or amino substituents on the naphthalimide chromophores bind at essentially identical sites. The footprinting profiles on tyrT DNA and on two fragments from pBS bear a remarkable resemblance to those determined for nogalamycin, an antibiotic which binds intercalatively leaving bulky carbohydrate substituents blocking both the major and minor grooves of the helix. Several lines of evidence indicate that the bis‐naphthalimides recognise their preferred binding sites via the unusual expedient of intercalating from the major groove. Footprints on the complementary DNA strands sometimes appear staggered in the 5′ direction. Repositioning the 2–amino group of G · C base pairs, which serves as a critical minor‐groove marker, by substitution with inosine and/or 2,6‐diaminopurine has little effect on the distribution of binding sites for LU 79553. The bisnaphthalimides affect the guanine‐specific reaction with dimethyl sulfate (which reacts with the N7 position of the base located in the major groove) but not reactions with tetrachloropalladinate or methylene blue. Photoactivation of LU 79553–DNA complexes leads to a small amount of strand scission mainly at guanine residues. These observations make a strong case for binding via the major groove of the double helix, in contrast to nearly all common intercalating drugs, which could be important in explaining the unique biological selectivity of bis‐naphthalimides.
Crystal Structure and Amino Acid Sequence of Wolinella Succinogenesl‐Asparaginase
Authors: Lubkowski Jacek , Palm Gottfried Julius , Gilliland Gary L. , Derst Christian , Röhm Klaus‐Heinrich , Wlodawer Alexander
Published: July 2004

The amino acid sequence and tertiary structure of Wolinella succinogenesl‐asparaginase were determined, and were compared with the structures of other type‐II bacterial l‐asparaginases. Each chain of this homotetrameric enzyme consists of 330 residues. The amino acid sequence is 40–50% identical to the sequences of related proteins from other bacterial sources, and all residues previously shown to be crucial for the catalytic action of these enzymes are identical. Differences between the amino acid sequence of W succinogenesl‐asparaginase and that of related enzymes are discussed in terms of the possible influence on the substrate specificity. The overall fold of the protein subunit is almost identical to that observed for other l‐asparaginases. Two fragments in each subunit, a very highly flexible loop (?20 amino acids) that forms part of the active site, and the N‐terminus (two amino acids), are not defined in the structure. The orientation of Thr14, a residue probably involved in the catalytic activity, indicates the absence of ligand in the active‐site pocket. The rigid part of the active site, which includes the asparaginase triad Thr93‐Lys166‐Asp94, is structurally very highly conserved with equivalent regions found in other type‐II bacterial l‐asparaginases.
Processing of Mammalian rRNA Precursors at the 3′ End of 18S rRNA
Authors: Cavaillé Jérôme , Hadjiolov Asen A. , Bachellerie Jean‐Pierre
Published: July 2004

Molecular mechanisms involved in the nucleolytic cleavage at the 18S rRNA/internal transcribed spacer 1 (ITS 1) junction, a late step of small‐subunit pre‐rRNA processing in vertebrates, remain largely unknown, mostly due to the lack of faithful in vitro assays. To identify the minimal cis‐acting signals required for this reaction, we studied the processing of truncated human rRNA gene transcripts transiently expressed upon transfection of rRNA minigenes into cultured mouse cells. We observed that processing at this site was faithfully reproduced with transcripts containing only 60 nucleotides of 18S rRNA and the adjacent 103 nucleotides of ITS 1, but was abolished or severely altered by further shortening of either sequence. Remarkably, this minimal transcript contains, within its 18S rRNA part, long sequences complementary to both U20 and U13 small nucleolar RNAs (snoRNAs). The cis‐acting elements essential for the reaction were studied further by site‐directed mutagenesis. The U20 snoRNA complementary region in 18S rRNA was not required for faithful processing at the 18S rRNA/ITS 1 junction. Also, processing at this site was not appreciably altered by random substitution of proximal ITS 1 sequences (including the 5′ terminal nucleotide) or of the terminal nucleotide of mature 18S rRNA. Substitutions in the four‐nucleotide loop of the 18S rRNA 3′‐terminal stem‐loop, including the two adenosine residues substrates of dimethylation, did not alter appreciably the formation of the 18S rRNA 3′ end, showing that the (methyl)2A1850 · (methyl)2A1851 doublet was not required for processing at this site. Two highly conserved 18S rRNA elements acted as major cis‐acting signals for processing at the 3′ end, the CAUU sequence immediately preceding the 3′‐terminal nucleotide and the 3′ strand of the 3′‐terminal 18S rRNA helix, complementary to U13 snoRNA. Compensatory mutations, restoring the potential for helix formation, but not U13 snoRNA complementarity, did not restitute the cleavage at the 3′ end of 18S rRNA. This suggests that U13 snoRNA may be a trans‐acting factor in the nucleolytic cleavage at the 3′ end of 18S rRNA.
Anion Binding to Mitochondrial Cytochromes c Studied through Electrochemistry
Authors: Battistuzzi Gianantonio , Borsari Marco , Dallari Daniela , Lancellotti Isabella , Sola Marco
Published: July 2004

The redox potential of horse and bovine heart cytochromes c determined through cyclic voltammetry is exploited to probe for anion–protein interactions, using a Debye–Hückel‐based model. In parallel, protein charge neutralization resulting from specific anion binding allows monitoring for surface‐charge/Eo relationships. This approach shows that a number of anions, most of which are of biological relevance, namely Cl‐, HPO2‐4, HCO‐3, NO‐3, SO2‐4, ClO‐4, citrate3‐ and oxalate2‐, bind specifically to the protein surface, often in a sequential manner as a result of the presence of multiple sites with different affinities. The binding stoichiometries of the various anions toward a given cytochrome are in general different. Chloride and phosphate appear to bind to a greater extent to both proteins as compared to the other anions. Differences in binding specificity toward the two cytochromes, although highly sequence‐related, are observed for a few anions. The data are discussed comparatively in terms of electrostatic and geometric properties of the anions and by reference to the proposed location and amino acid composition of the anion binding sites, when available. Specific binding of this large set of anions bearing different charges allows the electrostatic effect on Eo due to neutralization of net positive protein surface charge(s) to be monitored. 1H NMR indeed indicates the absence of significant salt‐induced structural perturbations, hence the above change in Eo is predominantly electrostatic in origin. A systematic study of protein surface‐charge/Eo relationships using this approach is unprecedented. Values of 15–25 mV (extrapolated at zero ionic strength) are obtained for the decrease in Eo due to neutralization of one positive surface charge, which are of the same order of magnitude as previous estimates obtained with either mutation or chemical modification of surface lysines. The effects of the anion‐induced decrease of net positive charge on Eo persist also at a relatively high ionic strength and add to the general effects related to the charge shielding of the protein as a whole due to the surrounding ionic atmosphere: hence the ionic strength dependence of the rate of electron transfer between cytochromes c and redox partners could also involve salt‐induced changes in the driving force.
Nucleoside‐Diphosphate Kinase from Streptomyces coelicolor
Authors: Brodbeck Michel , Rohling Annette , Wohlleben Wolfgang , Thompson Charles J. , Süsstrunk Urs
Published: July 2004

Nucleoside‐diphosphate (NDP) kinase was purified from crude extracts of Streptomyces coelicolor to over 90% homogeneity in a single step using an adenosine 3′,5′‐cyclic monophosphate (cAMP) binding column. The specific activity of protein in the fraction eluted with cAMP (400 U/mg) was about 3600‐fold higher than that in the crude extract. This enzyme was autophosphorylated in the presence of [γ‐32P]ATP. The high‐energy phosphoenzyme intermediate was stable in alkali and highly labile in acid; this suggests the presence of an N‐phosphate amino acid (most probably a histidine residue). A tetrameric form of the 15‐kDa protein was suggested by its apparent molecular mass (66 kDa) on a gel filtration column. The measured Michaelis constant (Km) for ATP was 85 μM. The IC50 for cAMP of 6 mM suggested weak competitive inhibition. However, no evidence that cAMP acts as an allosteric effector was obtained. The ndk gene from S. coelicolor was isolated and sequenced. The deduced amino acid sequence was very similar to other NDP kinases. However, unique characteristics were also noted, including a truncaled C‐terminus that makes it one of the smallest NDP kinases reported in the literature.
Structural Comparison of the Precursor and the Mature Form of Napin, the 2S Storage Protein in Brassica napus
Authors: Murén Eva , Ek Bo , Björk Ingemar , Rask Lars
Published: July 2004

The 2S storage protein napin from Brassica napus var. L. is synthesised as a precursor protein at the endoplasmic reticulum and transported along a gradient of decreasing pH to the vacuole, where two propeptides are removed to produce mature napin. The structures of pronapin expressed in insect cells and mature napin from rape seed were characterised. Limited proteolysis with several endoproteases cleaved primarily in the propeptides, suggesting that the propeptides are exposed to the exterior of the protein. Immunological comparison in parallel with circular dichroic spectrometry, both at neutral and acid pH, indicated that the propeptides had only a minor influence on the conformation of the regions of the molecule that correspond to mature napin.
The Use of Photolabelled Peptides to Localize the Substance‐P‐Binding Site in the Human Neurokinin‐1 Tachykinin Receptor
Authors: Girault Sophie , Sagan Sandrine , Bolbach Gérard , Lavielle Solange , Chassaing Gérard
Published: July 2004

The amino acid p ‐benzoyl‐l‐phenylalanine, (p ‐Bz)Phe, has been incorporated into substance P (SP), Arg‐Pro‐Lys‐Pro‐Gln‐Gln‐Phe‐Phe‐Gly‐Leu‐Met‐NH2, to localize the agonist‐binding domains of the human neurokinin‐1 (NK‐1) receptor overexpressed in a transfected mammalian cell line. The NK‐1‐specific agonist [Pro9]SP was modified at position 8 by (p ‐Bz)Phe and acylated at the N‐terminus by a biotinyl sulfone reporter via a 5‐aminopentanoyl spacer. After photolysis, the biotinyl sulfone moiety allowed easy and efficient removal of biotinylated fragments from the complex incubation mixture with streptavidin‐coated beads. Direct elution from the beads with the matrix used for matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS), which was facilitated by saturation of streptavidin sites with biotin, and subsequent MALDI‐TOF mass spectrometry analysis allowed identification of the NK‐1 fragments obtained after photolysis and proteolytic digestion. Trypsin digestion and combined trypsin/Staphylococcus aureus V8 protease enzymatic cleavage established that the site of covalent attachment of the photolabelled SP resides in the second extracellular loop, Thr173–Arg177. Cyanogen bromide cleavage shows that the probe is covalently attached to the methyl group of a methionine residue from human NK‐1. These experiments identified Met174 as the modified residue.
Differences in the Magnesium Dependences of the Class I and Class II Aminoacyl‐tRNA Synthetases from Escherichia coli
Authors: Airas R. Kalervo
Published: July 2004

The magnesium dependences of the ATP/PPi exchange and tRNA aminoacylation of reactions were measured for six aminoacyl‐tRNA synthetases (isoleucyl‐, tyrosyl‐ and arginyl‐tRNA synthetases from class I, and histidyl‐, lysyl‐ and phenylalanyl‐tRNA synthetases from class II). The measured values were subjected to best‐fit analyses using sum square error calculations between the data and the calculated curves in order to find the mode of participation of the Mg2+ and to optimize the sets of the kinetic constants.

The following four dependences were observed: the class II synthetases require three Mg2+ for the activation reaction (including the one in MgATP), but the class I synthetases require only one Mg2+ (in MgATP); in class II synthetases both MgPPi and Mg2PPi participate in the pyrophosphorolysis of the aminoacyl adenylate. Arginyl‐tRNA synthetase from class I also shows a better fit if also Mg2PPi reacts, but in the isoleucyl‐ and tyrosyl‐tRNA synthetases only MgPPi but not Mg2PPi is used in the pyrophosphorolysis. Different synthetases have different requirements for the tRNA‐bound Mg2+ and spermidine, independent of the enzyme class. 1–4 Mg2+ or spermidines are required in the best fit models. At the end of the reaction in all the synthetases analysed the dissociation of Mg2+ from the product aminoacyl‐tRNA essentially enhances the subsequent dissociation of the aminoacyl‐tRNA from the enzyme. The binding of ATP to the E · aminoacyl‐tRNA complex also speeds up the dissociation of the aminoacyl‐tRNA from most of these enzymes.
Heterologous Expression and Site‐Directed Mutagenesis of the 1‐Aminocyclopropane‐1‐Carboxylate Oxidase from Kiwi Fruit
Authors: Lay Venetia J. , Prescott Andy G. , Thomas Paul G. , John Philip
Published: July 2004

A system has been developed for the expression in Escherichia coli of 1‐aminocyclopropane‐1‐carboxylate (ACC) oxidase from kiwi fruit. In this first report of site‐directed mutagenesis of ACC oxidase, seven different mutants of the enzyme have been expressed, and their activities compared to that of the heterologously expressed wild‐type enzyme. No great loss of activity was observed when Lys172 was substituted by either Ala or Cys, or when Gly137 was substituted by Pro. However, the mutant proteins showed only 1% of the wild‐type activity when substitutions were made of Asp179, His177, and Lys158. The results are discussed in terms of possible mechanisms by which ACC oxidase is activated by carbon dioxide, and in terms of structural motifs suggested by the known structure of isopenicillin N‐synthase, an enzyme related by mechanism and sequence similarity to ACC oxidase. It is concluded that Lys172, a putative carbon dioxide binding site, has no role to play in the catalytic activity of the enzyme. The results support a previous suggestion that ACC oxidase shares important structural features with isopenicillin N‐synthase.