bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical and biophysical analysis of pseudoknot‐containing RNA fragments
Authors: van BELKUM Alex , WIERSEMA Pieter Jan , JOORDENS Jos , PLEIJ Cornelis , HILBERS Cornelis W. , BOSCH Leendert
Published: March 2005

Three overlapping RNA fragments containing the pseudoknot, as found in the tRNA‐like structure of turnip yellow mosaic virus (TYMV) RNA, have been isolated and purified. Site‐directed cleavage of TYMV RNA by RNase H, followed by ammonium sulphate precipitation and ion‐exchange HPLC, yielded a pure preparation of a 3′‐terminal, 112‐nucleotide TYMV RNA fragment. Transcription of TYMV cDNA by T ‐ RNA polymerase, resulted in the isolation of an 88‐nucleotide fragment. Finally, a 44‐nucleotide fragment containing the TYMV RNA pseudoknot and strongly resembling the aminoacyl acceptor arm of the viral RNA was also synthesised using T7 RNA polymerase. The three fragments were isolated in milligram amounts and used for biochemical structure mapping, ultraviolet melting studies and NMR spectroscopy.

Chemical modification with diethyl pyrocarbonate and sodium bisulphite and enzymatic digestion with RNase T1 confirmed the presence of the pseudoknot in the 44‐nucleotide fragment. Also the analogue of the T‐stem and T‐loop of the tRNA‐like structure of TYMV RNA was found. The results of modification at various temperatures in Mg2+ ‐containing buffers were in general agreement with optical melting studies. Ultraviolet melting analysis of the longer fragments revealed their greater complexity and the results appear similar to those obtained for some tRNA species.

To obtain direct biophysical evidence for base‐pairing and stacking interactions in the pseudoknot. NMR studies were initiated. The first proton‐NMR spectra ever obtained for plant viral RNA fragments are presented. NMR spectra were recorded at various buffer conditions and at various temperatures. The spectra for the 112‐nucleotide and 88‐nucleotide fragment are too complicated to be solved at present. In the case of the 44‐nucleotide fragment, however, the imino proton resonances are well separated and this system turns out to be most promising for structural studies.
Investigation of RecA—polynucleotide interactions from the measurement of LexA repressor cleavage kinetics
Authors: TAKAHASHI Masayuki , SCHNARR Manfred
Published: March 2005

The proteolysis of the LexA repressor in the presence of RecA and various polynucleotides was studied by measuring the fluorescence decrease of LexA upon cleavage. The results were compared with the DNA binding of RecA to invéstigate the presence of multiple DNA‐RecA complexes. All single‐stranded polydeoxyribonucleotides (DNA) efficiently stimulated the proteolysis and the maximum activation was reached in the presence of three or four nucleotides of polynucleotide per monomer of RecA. The stimulative effect was decreased in the presence of larger amounts of poly(dA), poly(dT) or heat‐denatured DNA, whereas the excess of single‐stranded DNAs chemically modified with chloroacetaldehyde did not present such an inhibitory effect, despite the fact that a second DNA molecule is likely to interact with RecA as monitored by the intrinsic fluorescence of these DNA species. The complicated cleavage promotion and inhibition pattern is tentatively explained by a three‐state model assuming that RecA may interact with three single‐stranded DNA molecules. According to this model, occupation of the first site would be necessary and sufficient for cleavage promotion, the second site would be neutral with respect to cleavage and the occupation of the third site would inhibit LexA cleavage at least partially. Double‐stranded natural DNA did not stimulate cleavage, even under conditions where RecA binds quantitatively to the DNA. No polyribonucleotides (RNA) examined showed a significant stimulative effect either. nor did they appear to interact with RecA.
Purification and characterization of trimming glucosidase I from pig liver
Authors: BAUSE Ernst , SCHWEDEN Jürgen , GROSS Achim , ORTHEN Bruno
Published: March 2005

Trimming glucosidase I has been purified about 400‐fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N‐glycoprotein as shown by its binding to concanavalin A—Sepharose and its susceptibility to endo‐β‐N‐acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non‐specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N‐linked high‐mannose sugar chain.

Purified pig liver glucosidase I hydrolyzes specifically the terminal α1–2‐linked glucose residue from natural Glc3‐Man9‐GlcNAc2, but is inactive towards Glc2‐Man9‐GlcNAc2 or nitrophenyl‐/methyl‐umbelliferyl‐α‐glucosides. The enzyme displays a pH optimum close to 6.4. does not require metal ions for activity and is strongly inhibited by 1‐deoxynojirimycin (Ki∼ 2.1 μM), N,N‐dimethyl‐1‐deoxynojirimycin (Ki∼ 0.5 μM) and N‐(5‐carboxypentyl)‐1‐deoxynojirimycin (Ki∼ 0.45 μM), thus closely resembling calf liver and yeast glucosidase I.

Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross‐react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross‐reactivity.
Preparation of biologically active platelet‐derived growth factor isoforms AA and AB
Authors: HOPPE Jürgen , WEICH Herbert A. , EICHNER Wolfram , TATJE Dieter
Published: March 2005

We have expressed the mature platelet‐derived growth factor (PDGF) A chain within a fusion protein of the cro repressor and β‐galactosidase in Escherichia coli. Monomeric PDGF‐A was excised from this fusion protein by CNBr cleavage. After protection of thiols by S‐sulfonation, this fragment was purified by gel permeation chromatography and reversed‐phase high‐performance liquid chromatography. The monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active recombinant AA dimer (rPDGF‐AA) with an overall yield of about 0.2 mg/l culture. When monomeric rPDGF‐A and rPDGF‐B were reacted at stoichiometric concentrations in the presence of glutathione, almost exclusively hetero‐dimers of type AB were formed. Heterodimers AB stimulated [3H]thymidine incorporation into AKR‐2B fibroblasts half‐maximally at about 2 ng/ml. AA homodimers were fivefold less active. About 60000 binding sites were found for rPDGF‐AB, 30000 for rPDGF‐AA and 45000 for rPDGF‐BB on AKR‐2B fibroblasts.
Occurrence of S‐(1,2‐dicarboxyethyl)‐cysteine at position 77 in mutant human lysozyme secreted by Saccharomyces cerevisiae
Authors: KIKUCHI Masakazu , TANIYAMA Yoshio , KANAYA Shigenori , TAKAO Toshifumi , SHIMONISHI Yasutsugu
Published: March 2005

A mutant human lysozyme P110, in which Val110 was replaced with Pro, was secreted by Saccharomyces cerevisiae; modification of the cysteine residue at position 77 was found in a purified mutant protein (P110‐B) upon primary structure analysis. A peptide fragment containing 15 amino acid residues from Thr70 to Leu84 was obtained by proteolytic digestion of the protein and subsequently isolated by reverse‐phase HPLC. This fragment was analyzed by high‐resolution fast‐atom‐bombardment (FAB) mass spectrometry, which showed that 1,2‐dicarboxyethyl group was attached to the thiol group of Cys77. This modification was confirmed by comparing it with a sample of chemically synthesized S‐(1,2‐dicarboxyethyl)‐L‐cysteine.

It was found that the modification caused a disruption of the disulfide bond Cys77–Cys95 in the mutant molecule. These observations, plus structural considerations, suggest that Cys77 and Cys95 either remain uncrosslinked or the disulfide bond Cys77–Cys95, once formed, is opened during the final step in the folding of human lysozyme in vivo.
Characterisation of the Klebsiella pneumoniae nitrogen‐fixation regulatory proteins NIFA and NIFL in vitro
Authors: AUSTIN Sara , HENDERSON Neville , DIXON Ray
Published: March 2005

Transcriptional activation by the Klebsiella pneumoniae nitrogen‐fixation‐specific positive control protein, NIFA, (nifA gene product) has been demonstrated in vitro in S30 extracts from cells which overproduce this protein. The activity of NIFA was dramatically reduced in vitro in the presence of the negative regulatory protein NIFL (nifL gene product) but was not inhibited by the presence of a mutant NIFL protein, NIFL2404. Transcriptional activation from the nifH promoter by NIFA was dependent on the alternative σ factor, σ54, and also required the presence of an upstream activator sequence. NIFA activity was temperature‐sensitive in vitro (as it is in vivo) which is due, at least in part, to the intrinsic lability of the protein itself.

The majority of overproduced NIFA and NIFL was insoluble after low‐speed centrifugation and was inactive in vitro. A low level of less aggregated NIFA protein present in cell extracts was responsible for in vitro activity and this fraction was partially purified.
Structural studies of the lysozyme coded by the pneumococcal phage Cp‐1
Authors: SANZ Jesús M. , GARCIA José L.
Published: March 2005

The CPL‐1 lysozyme coded by the pneumococcal phage Cp‐1 has been overproduced in Escherichia coli under the control of a modified lipoprotein lactose promoter. This result has provided the conditions to analyse the CPL‐1 secondary structure by circular dichroism (CD). The CD spectra recorded in the far‐ultraviolet region showed, at neutral pH, two minima at 210 nm and 230 nm and a shoulder at 217 nm, whereas two bands at 260 nm and 295 nm were observed in the near‐ultraviolet region. It has been estimated, by using the CDPROT program, that the protein is composed of 19%α‐helix, 32%β‐sheet, 28%β‐turn and 21% random coil. Minor changes in the CD spectra were detected either when the pH was varied over 6–10 or when the ionic strength was increased to 1 M NaCl. Choline, a well known modulator of the enzyme activity that is present in the pneumococcal cell wall, induced remarkable changes in the intensities of the bands at 210, 230 and 295 nm, with the appearance of an unusual positive band at 225 nm. The conformational change was reversible and correlated with the competitive inhibitory effect of choline on the lysozyme activity, supporting, by a new and direct experimental approach, the basic role of choline in the recognition of the cell wall substrate. The analyses of the secondary structure prediction and the CD data reported here are compatible with the two‐domain structure of CPL‐1 reinforce our hypothesis that the C‐terminal region is directly involved in the binding of the enzyme to the pneumococcal teichoic and lipoteichoic acids.
De novo fatty acid synthesis mediated by acyl‐carrier protein in Neurospora crassa mitochondria
Authors: MIKOLAJCZYK Steve , BRODY Stuart
Published: March 2005

The acyl‐carrier protein (ACP) in Neurospora crassa mitochondria [Brody, S. & Mikolajczyk, S. (1988) Eur. J. Biochem. 173, 353–359] mediated a cerulenin‐sensitive, de novo fatty acid synthesis independent of the fatty acid synthetase complex present in the cytoplasm. Incubation of mitochondria with [2‐14C]malonate labeled only the ACP as indicated by autoradiography after SDS/PAGE. Under these in vitro conditions ATP was required for the initial acyl‐ACP formation, but further elongation required either magnesium or the direct addition of NADPH. Labeled hexanoic (6:0) and caprylic (8:0) acids were detected as intermediates in the pathway, as well as hydroxymyristic acid. All of the intermediates, and the eventual product of the reaction, myristic acid (14:0), were released from the ACP by alkaline treatment. Pulse‐chase experiments demonstrated the incorporation on to, and release of label from, the ACP. In vivo labeling of ACP with [2‐14C]malonate was also detected and the label was in the form of hydroxymyristic acid. This newly discovered pathway is discussed from the standpoint of its possible role in providing acyl chains for mitochondrial lipids.
Purification and characterization of a novel β‐agarase from Vibrio sp. AP‐2
Authors: AOKI Takahiko , ARAKI Toshiyoshi , KITAMIKADO Manabu
Published: March 2005

β‐Agarase was purified from the culture fluid of a porphyran‐decomposing marine bacterium (strain AP‐2) by ammonium sulfate precipitation, successive column chromatography and DNase and RNase treatment. The final enzyme preparation appeared to be homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 20 kDa, a pH optimum of 5.5, and was stable in the pH region 4.0–9.0 and at temperatures below 45°C.

The β‐agarase was a novel endo‐type enzyme which hydrolyzed neoagarotetraose, larger neoagarooligosaccharides and agar to give neoagarobiose [3,6‐anhydro‐α‐L‐galactopyranosyl‐(1→3)‐D‐galactose] as the predominant product. The enzyme did not act on K‐carrageenan.

According to the criteria of Bergey's Manual of Systematic Bacteriology, the strain was assigned to the genus Vibrio.
Sequential 1H‐NMR assignments and secondary structure of the sea anemone polypeptide anthopleurin‐A
Authors: MABBUTT Bridget C. , NORTON Raymond S.
Published: March 2005

The sequence‐specific assignment of resonances in the 500‐MHz 1H‐NMR spectrum of a cardioactive sea anemone polypeptide, anthopleurin‐A, is described. The assignment procedure involved analysis of two‐dimensional phase‐sensitive multiple‐quantum‐filtered, double‐quantum, homonuclear Hartmann‐Hahn and nuclear Overhauser effect spectra. Using sequential information, specific assignments have been made for resonances arising from all 49 amino acid residues. Resonances arising from a number of residues in a minor conformer present in solution are also assigned. These results greatly extend previous resonance assignments made from spectra acquired at 300 MHz [Gooley, P. R. and Norton, R. S. (1985) Eur. J. Biochem. 153, 529–539] and provide the basis for a more accurate definition of the conformation of anthopleurin‐A in aqueous solution. The secondary structure includes a four‐stranded antiparallel β‐sheet encompassing residues 2–4, 21–23, 34–36 and 45–49, and possibly a β‐bulge located at Ser‐19 and Gly‐20. A type II β‐turn is formed by residues 30–33. These structural elements also occur within other related sea anemone polypeptides, but the conformation of the small loop region containing Pro‐41 appears to be unique to anthopleurin‐A.
δ‐Haemolysin from Staphylococcus aureus and model membranes
Authors: DUFOURC Erick J. , DUFOURCQ Jean , BIRKBECK Thomas H. , FREER John H.
Published: March 2005

Solid‐state 2H NMR and 31P NMR of 2H‐enriched chains and polar head groups, respectively, of dipalmitoylglycerophosphatidylcholine/water dispersions were undertaken to investigate the action of δ‐haemolysin from Staphylococcus aureus on biomembranes. When the lipid/toxin molar ratio, Ri is 10, the gelphase 2H powder patterns and the temperature of the gel–fluid phase transition, tc, are unchanged by the presence of the toxin whereas the 31P powder spectra of polar head groups are perturbed. At t > tc, a detailed analysis of methylene ordering indicates that δ‐haemolysin orders the lipid chains near tc and disorders them for t≫tc. These findings are interpreted in terms of peptide location with regard to the membrane and suggest that the position of the toxin depends on the temperature relative to tc.

Experiments carried out at Ri= 4 exhibit sharp, isotropic 2H‐NMR lines, at t > tc, indicating that δ‐haemolysin promotes the appearance of very small objects undergoing fast isotropic reorientation which average to zero the quadrupolar interaction. Below tc, one observes gel‐phase powder patterns which indicate that the bacterial toxin is unable to form such small structures with ordered dipalmitoylglycerophosphocholine phospholipids. From comparison of the action of δ‐haemolysin with that of melittin on same lipids [Dufourc et al. (1986) Biochemistry 25, 6448–6455] it results that both toxins perturb similarly fluid‐phase lipids at elevated temperature, but they behave differently with gel‐phase lipids, the former toxin being less efficient in membrane restructuring than the latter.
Functional and immunological evidence for stable association of solubilized vasoactive‐intestinal‐peptide receptor and stimulatory guanine‐nucleotide‐binding protein from rat liver
Authors: COUVINEAU Alain , ROUYER‐FESSARD Christiane , VOISIN Thierry , LABURTHE Marc
Published: March 2005

We have reported the solubilization of complexes between vasoactive intestinal peptide (VIP) and its receptor from rat liver in a GTP‐sensitive form of Mr 150 000 [Couvineau, A., Amiranoff, B. & Laburthe, M. (1986) J. Biol. Chem. 261, 14 482–14 489]. In the present study, we demonstrate a stable association of solubilized VIP receptor and stimulatory guanine nucleotide‐binding protein (Gs protein), taking advantage of the ability of the glycoproteic VIP receptor (Mr 48 000), and the inability of the Gs protein, to adsorb to wheat germ agglutinin (WGA). 125I‐VIP‐receptor complexes solubilized in Triton X‐100 were adsorbed on WGA‐Sepharose, extensively washed and the radioactivity retained was eluted with 1 mM GTP showing that: (a) radioactivity corresponds to free 125I‐VIP and (b) αs (Mr 42 000) and β (Mr 35 000) subunits of Gs protein are detectable in the GTP eluate by immunoblotting using antisera against these subunits. Such an effect of GTP implied that a stable ternary complex consisting of VIP, receptor and Gs protein had been adsorbed to WGA‐Sepharose. When Triton‐solubilized 125I‐VIP‐receptor complexes were adsorbed on WGA‐Sepharose, then retained material was specifically eluted with 0.3 M N‐acetylglucosamine, analysis of the sugar eluate showed the following results. (a) GTP induces the dissociation of 125I‐VIP‐receptor complexes of Mr 150 000 contained in the eluate indicating that 125I‐VIP‐receptor‐G protein complexes had been adsorbed to the WGA column. (b) The Mr‐42 000 αs subunit can be specifically ADP‐ribosylated by cholera toxin. (c) Immunoblotting using antisera against the αs and β subunits of Gs protein, reveals Mr‐42 000 and Mr‐35 000 components corresponding to αs and β subunits, respectively. (d) Affinity cross‐linking using dithiobis(succinimidyl‐propionate) of 125I‐VIP‐receptor complexes eluted from the WGA column reveals a major band corresponding to Mr 150 000. Immunoblotting using antisera against the β‐subunit shows the presence of the β subunit (Mr 35 000) in this Mr‐150 000 component.

In conclusion, these data provide functional and immunochemical evidence for the physical association of solubilized VIP‐receptor complexes with αs and β subunits of Gs protein.
The proline‐activating activity of the multienzyme gramicidin S synthetase 2 can be recovered on a 115‐kDa tryptic fragment
Authors: SKARPEID Hans‐Jacob , ZIMMER Trine‐Lise , SHEN Beifen , von DÖHREN Hans
Published: March 2005

The multienzyme gramicidin S synthetase 2 was treated with trypsin to obtain fragments capable of activating proline. Three different active fragments were detected. The course of proteolysis was simulated by using a concentration range of trypsin; the cleavage pattern indicated that one of the fragments was particularly stable. This fragment was purified and shown to have a molecular mass of 115 kDa. It was compared chromatographically, by SDS/PAGE, and enzymatically to a Pro‐activating fragment produced by a gramicidin‐S‐negative mutant. It can be concluded that the proteolytic fragment represents a structure which is contained on a continuous part of the polypeptide chain of gramicidin S synthetase 2 and has a relatively compact structure. This provides evidence that the multienzyme gramicidin S synthetase 2 is, at least in part, constructed from functional domains. An approach towards extending these studies to other parts of the gramicidin S synthetase 2 molecule has also been devised. This work complements recombinant DNA studies in the area, providing stable functional fragments.
1H‐NMR and mass spectrometric studies of tetrahydropterins
Authors: GHISLA Sandro , KUSTER Thomas , STEINERSTAUCH Petra , LEIMBACHER Walter , RICHTER Wilhelm Johannes , RASCHDORF Fritz , DAHINDEN Roland , CURTIUS Hans‐Christoph
Published: March 2005

The conversion of dihydroneopterin triphosphate in the presence of 6‐pyruvoyl tetrahydropterin synthase was followed by 1H‐NMR spectroscopy. The interpretation of the spectra of the product is unequivocal: they show formation of a tetrahydropterin system carrying a stereospecifically oriented substituent at the asymmetric C(6) atom. The spectra are compatible with formation of a (3′)‐CH3 function, and with complete removal of the 1′ and 2′ hydrogens of dihydroneopterin triphosphate. The fast‐atom‐bombardment/mass spectrometry study of the same product yields a [M + H]+ ion at m/z 238 compatible with the structure of 6‐pyruvoyl tetrahydropterin. The data support the proposed structure of 6‐pyruvoyl tetrahydropterin as a key intermediate in the biosynthesis of tetrahydrobiopterin.
Structure of preproattacin and its processing in insect cells infected with a recombinant baculovirus
Authors: GUNNE Hans , HELLERS Marianne , STEINER Håkan
Published: March 2005

From a cDNA library made from fatbody of immunized Hyalophora cecropia pupae, a full‐length clone corresponding to basic attacin was isolated. The corresponding amino acid sequence suggests that basic attacin is synthesized as a 233‐residue preproprotein. This was confirmed by cloning the cDNA fragment encoding the basic attacin in the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter and expressing the protein in Spodoptera frugiperda (Sf9) cells. Biologically active attacin was also produced in last instar larvae of Trichoplusia ni after injection of recombinant virus. The concentration obtained was 300–500 times that obtained in cell culture supernatants. In cell culture the induction kinetics of attacin were followed at both transcriptional and translational levels. From the protein processing pattern it was suggested that a protease produced by the Spodoptera cells cleaves attacin at the double arginine residues at positions 45 and 46. The instability of the attacin proteins is rationalized in terms of their random‐coil structure which was deduced from circular dichroism measurements.
Arrangement of disulfide bridges and positions of sulfhydryl groups in tetanus toxin
Authors: KARIEGLSTEIN Kerstin , HENSCHEN Agnes , WELLER Ulrich , HABERMANN Ernst
Published: March 2005

Tetanus toxin is a 151‐kDa protein. The complete amino acid sequence is known. The mature toxin is made up of the two peptide chains and contains 10 half‐cystine residues. Treatment with 4‐vinylpyridine in the presence of 6 M guanidine converted six of them into S‐pyridylethyl cysteine residues as determined by amino acid analysis. When alkylation was preceded by mercaptolysis, all 10 half‐cystine residues were recovered in the S‐pyridyl‐ethylated form. It was therefore concluded that the toxin contains six sulfhydryl groups and two disulfide bonds.

The positions of the residues carrying sulfhydryl groups and of those involved in disulfide bridges were determined by labelling of the toxin alternatively with 4‐vinylpyridein or with 4‐dimethylaminoazobenzene‐4′‐iodoacetamide (DABIA), directly or after mercaptolysis. The toxin derivatives were cleaved with cyanogen bromide and the elution patterns in reversed in reversed‐phase HPLC compared. The chromatography components were identified by N‐terminal amino acid sequence and amino acid composition. In the chromatography of the non‐mercaptolysed, DABIA‐treated sample four chromophore‐carrying components were detected which could be demonstrated by N‐terminal sequence analysis to correspond to six half‐cystine‐containing cyanogen bromide fragments. In the mercaptolysed, DABIA‐treated sample three additional chromophore‐carrying components were present, corresponding to two previously disulfide‐linked cyanogen bromide fragments and one fragment which had contained an internal disulfide bridge. The HPLC patterns showed characteristic differences as the DABIA‐labelled fragments were considerably more hydrophobic than the corresponding vinylpyridine‐labelled fragments.

It was established that the half‐cystine residues in positions 26, 185, 198, 311, 868, and 1300 are present in the sulfhydryl form, that those in positions 438 and 466 are disulfide‐bridged, thereby connecting the light and heavy chains of the toxin, and that those in positions 1076 and 1092 are disulfide‐bridged, thereby giving rise to a loop in the heavy chain. During the progress of the investigations about 20% of the amino acid sequence previously predicted from DNA analysis was confirmed by protein‐chemical methods.
Use of fragments of hirudin to investigate thrombin‐hirudin interaction
Authors: DENNIS Stanley , WALLACE Andrew , HOFSTEENGE Jan , STONE Stuart R.
Published: March 2005

Site‐directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C‐terminal fragment (residues 53–65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D‐phenylalanylpipecolylarginyl‐p‐nitroaniiide (dPhe‐Pip‐Arg‐NH‐Ph). The N‐terminal fragment (residues 1–52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C‐terminal fragment protected α‐thrombin against cleavage by trypsin, the N‐terminal fragment did not. Hirudin and the N‐terminal fragment completely prevented the cleavage of α‐thrombin by pancreatic elastase while the C‐terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.
Metabolic studies with NMR spectroscopy of the alga Dunaliella salina trapped within agarose beads
Authors: BENTAL Michal , PICK Uri , AVRON Mordhay , DEGANI Hadassa
Published: March 2005

A technique for the entrapment of the ünicellular algae Dunaliella salina in agarose beads and their perfusion during NMR measurements is presented. The trapped cells maintained their ability to proliferate under normal growth conditions, and remained viable and stable under steady‐state conditions for long periods during NMR measurements. Following osmotic shock in the dark, prominent changes were observed in the intracellular level of ATP and polyphosphates, but little to no changes in the intracellular pH or orthoposphate content. When cells were subjected to hyperosmotic shock, the ATP level decreased. The content of NMR‐visible polyphosphates decreased as well, presumably due to the production of longer, NMR‐invisible structures. Following hypoosmotic shock, the ATP content increased and longer polyphosphates were broken down to shorter, more mobile polymers.
Ontogenesis of rabbit liver cytochrome P450
Authors: BONFINS Claude , COMBALBERT Jean , PINEAU Thierry , ANGEVIN Jacques , LARROQUE Christian , DERANCOURT Jean , CAPONY Jean‐Paul , MAUREL Patrick
Published: March 2005

The liver hydroxylating system, mainly composed of cytochromes P450, is not highly active during foetal life. If develops after birth and reaches the adult level several weeks post‐partum. We have studied the ontogenesis of rabbit cytochrome P450 during the post‐natal period. Total P450 as well as isozymes 2, 3b, 3c, 4 and 6 were measured. The evolution of these proteins with ageing, together with qualitative modification of an electrophoretic profile, produced evidence of an early developing P450 prevailing from one week to three weeks after birth. We isolated and characterized a cytochrome, called P450 2y, from two‐week liver microsomes. It is closely related to P450450 3a, an adult form of rabbit P450 induced by ethanol. They have similar molecular masses, the same λmax of CO‐reduced spectrum and exhibit immunological cross‐reactivity. However, we cannot conclude that the two proteins are identical from N‐terminal amino acid analysis or the two‐dimensional gel electrophoresis pattern. These results, as well as the recent evidence of two different genes coding for the P450 3a family, strengthen the idea that P450 2y and 3a are distinct proteins, P450 2y seems to be an early developing form abundant soon after birth, while P450 3a is a delayed form appearing like most P450 isozymes during the fourth post‐natal week. Besides the quantitative development during perinatal life, there is an important qualitative modification of liver cytochrome P450 content.
Asymmetric distribution of the phosphatidylinositol‐linked phospho‐oligosaccharide that mimics insulin action in the plasma membrane
Authors: VARELA Isabel , ALVAREZ José F. , CLEMENTE Rosa , RUIZ‐ALBUSAC Juan M. , MATO José M.
Published: March 2005

We have investigated the topography of a glycosyl‐phosphatidylinositol implicated in insulin action by a combination of two complementary methods: (a) chemical labelling with a non‐permeable (isethionyl acetimidate) and a permeable (ethyl acetimidate) probe; and (b) enzymatic modifications with β‐galactosidase (EC 3.2.1.23) or phosphatidylinositol‐specific phospholipase C (EC 3.1.4.3). Using the first approach the majority of the glycosyl‐phosphatidylinositol is found in the outer surface of intact hepatocytes, adipocytes, fibroblasts and lymphocytes, but not in erythrocytes which presented only a 20% of the total labelled glycosyl‐phosphatidylinositol to the exterior. Upon insulin addition (10 nM), about 60% of the total glycosyl‐phosphatidylinositol was hydrolysed in both hepatocytes and adipocytes but not in erythrocytes. In agreement with the extracellular localization in hepatocytes and with the proposed role of this glycolipid in insulin action, treatment of rat hepatocytes with β‐galactosidase from Escherichia coli, an enzyme that hydrolyses the oligosaccharide moiety of the glycosyl‐phosphatidylinositol, cleaved 65% of the total glycophospholipid and blocked the effect of insulin (but not of glucagon) on pyruvate kinase (EC 2.7.1.40). Similar treatment with phosphatidylinositol‐specific phospholipase C from Bacillus cereus hydrolysed 62% of the total glycosyl‐phosphatidylinositol. From the various approaches used it is concluded that the majority of this glycophospholipid is at the outer surface in a variety of insulin‐sensitive cells.
Fast and slow isoforms of troponin I and troponin C
Authors: HÄRTNER Karl‐Thomas , PETTE Dirk
Published: March 2005

Polyclonal antibodies were raised against troponin I (TnI) and troponin C (TnC) purified from fast‐twitch and slow‐twitch rabbit muscles. These antibodies were used to elucidate the distribution of fast and slow isoforms of TnI and TnC in normal and chronically stimulated rabbit hind limb muscles by immunoblots of one‐dimensional and two‐dimensional electrophoreses. In contrast to the multiplicity of fast and slow troponin T (TnT) isoforms, TnI and TnC were present as unique fast and slow isoforms. Whereas no charge variants were detected for slow TnI, fast TnI was present in at least three charge variants. As judged from the results of alkaline phosphatase digestion, these charge variants represent differently phosphorylated forms. Fast and slow TnC both exist as two charge variants which, however, were unaffected by alkaline phosphatase treatment. Chronic low‐frequency stimulation of fast‐twitch muscles induced progressive increases in the slow isoforms of TnC and TnI at the expense of their fast isoforms. The extent of the fast‐to‐slow transition was more pronounced in the case of TnC than in that of TnI. Long‐term stimulated muscles with a complete fast‐to‐slow transition, at the level of the TnT isoforms, still contained fast and slow isoforms of both TnI and TnC. The coexistence of fast and slow isoforms of the three troponin subunits in the transforming muscle was interpreted as indicating the presence of hybrid troponin molecules composed of fast and slow isoforms. Studies at the mRNA level showed changes similar to those at the protein level. However, in long‐term stimulated muscles, the fast‐to‐slow transition of TnI was more pronounced at the mRNA level than at the protein level.
Determination of the DNA conformation of the simian virus 40 (SV40) enhancer in SV40 minichromosomes
Authors: MARTÍNEZ‐BALBÁS Angeles , CASASNOVAS Jose María , AZORÍN Fernando
Published: March 2005

The simian virus 40 (SV40) enhancer contains three 8‐bp purine‐pyrimidine alternating sequences which are known to adopt the left‐handed Z‐DNA conformation in vitro. In this paper, we have undertaken the determination of the DNA conformation adopted by these Z‐motifs in the SV40 minichromosome. We have analyzed the presence of Z‐DNA through the change in linkage which should accompany formation of this left‐handed conformation. Our results indicate that, regardless of the precise moment of the viral lytic cycle at which minichromosomes are harvested and the condition of the transfected DNA, either relaxed or negatively supercoiled, none of the three Z motifs of the SV40 enhancer exist to a significant extent as Z‐DNA in SV40 minichromosomes. The SV40 enhancer adopts predominantly a right‐handed B‐DNA conformation in vivo.
A ‘top‐down’ approach to the determination of control coefficients in metabolic control theory
Authors: BROWN Guy C. , HAFNER Roderick P. , BRAND Martin D.
Published: March 2005

A new approach to the determination of flux and concentration control coefficients in metabolic pathways is outlined. Linear pathways are conceptually divided in two around an intermediate metabolite (or group of metabolites) and the control coefficients of the two parts are derived from the elasticity coefficients of the two parts to the intermediate. Branched pathways are treated similarly, the control coefficients of the branches being derived either from the elasticities of the branches to their common intermediate or from the relative flux changes of the branches. Repeating this analysis around other intermediates in the pathway allows the control coefficients of smaller and smaller groups of enzymes to be determined. In complex systems this approach to describing control may have several advantages over determining the control coefficients of individual enzymes and is a potentially useful complementary approach.
The diacylglycerol kinase inhibitor, R59022, potentiates cholecystokinin‐induced enzyme secretion from rabbit pancreatic acini
Authors: EDERVEEN Antoine G. H. , van EMST‐DE VRIES Sjenet E. , DE PONT Jan Joep H. H. M. , WILLEMS Peter H. G. M.
Published: March 2005

The putative inhibitor of diacylglycerol kinase activity, 6‐{2‐[(4‐fluorophenyl)phenylmethylene]‐1‐piperidinyl}‐ethyl‐7‐methyl‐5H‐thiazolo[3,2‐a]pyrimidin‐5‐one (R59022), markedly potentiated cholecystokinin‐C‐terminal‐octapeptide(CCK‐8‐)stimulated enzyme secretion from isolated rabbit pancreatic acini. Maximal potentiation occurred when acini were stimulated in the presence of 5–10 μM R59022. Potentiation depended both on the concentration of R59022 and CCK‐8. No potentiation was observed when acini were half‐maximally stimulated, whereas the secretory response to maximal and supramaximal concentrations of secretagogue was increased by 50–60%. R59022 alone had no effect on basal enzyme secretion and the drug did not potentiate the secretory response to the Ca2+ ionophore A23187 or to the phorbol ester, 12‐O‐tetradecanoylphorbol 13‐acetate. Moreover, no increase in basal secretion was observed when acini were incubated in the presence of both R59022 and forskolin. These observations strongly suggest that receptor‐mediated activation of the inositol phospholipid pathway is required for R59022‐induced potentiation. R59022‐inhibited the CCK‐8‐stimulated incorporation of 32Pi into phosphatidic acid dose dependently, without affecting the CCK‐8‐stimulated hydrolysis of 32P‐labelled phosphatidylinositol 4,5‐bisphosphate. This is consistent with an inhibitory effect of R59022 on acinar cell diacylglycerol kinase activity. The potentiating effect of R59022 was mimicked by 12‐O‐tetradecanoylphorbol 13‐acetate added simultaneously with CCK‐8.

Therefore, it is concluded that in the presence of 5–10 μM R59022 the receptor‐mediated increase in acinar cell diacylglycerol content is enhanced leading to enhanced activation of protein kinase C and to potentiation of the secretory response. The fact that the secretory response to maximal and supramaximal concentrations of CCK‐8 is potentiated by R59022 suggests that at these concentrations of secretagogue the diacylglycerol protein kinase C branch of the signal‐transduction route is rate‐limiting.
Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O–55Mn superhyperfine interaction
Authors: KALBITZER Hans Robert , FEUERSTEIN Jürgen , GOODY Roger S. , WITTINGHOFER Alfred
Published: March 2005

Electron paramagnetic resonance spectroscopy has been used to obtain information on the structure and stability of the products of GTP cleavage at the active site of elongation factor Tu (EF‐Tu) from Bacillus stearothermophilus. Using stereospecifically labelled (Sp)‐(Rp)‐[β‐17O]GTP (prepared by modification of a previously published procedure which is now also suitable for guanine nucleotides), it was found that only one of the two possible diastereomers (Sp) led to detectable line‐broadening of the EPR spectrum of Mn2+ at the active site of EF‐Tu (linewidth 1.5 mT), whereas the Rp isomer caused the same linewidth as unlabelled nucleotide (1.3 mT). From our earlier work and from a demonstration that the lifetime of the state giving the broadened spectrum is too long to be assigned to the EF‐Tu · GDP · Mn complex [the rate constant for decay as measured by displacement of GDP by the fluorescent 2′(3′)‐O‐(N‐methylanthraniloyl)‐GDP is 6.2 × 10−3s−1 at 25°C and pH 6.8], we conclude that the broadened signal arises from the EF‐Tu · Mn · GDP · Pi complex, the predominant steady‐state species. During the hydrolysis of GTP the Mn2− remains bound to the β‐phosphate oxygen of GDP which arises from the βpro‐S oxygen of GTP, possibly until GDP dissociates and certainly until Pi dissociates. Addition of elongation factor Ts (EF‐Ts) to this intermediate leads to rapid reduction of the linewidth to that expected for random distribution of interactions of one 17O and two 16O atoms of GDP with Mn2+, and is not distinguishable from that exhibited by (Rp)‐[β‐17O]GTP in the corresponding complex in the presence of EF‐Ts.
Topological analysis of hydrogen bonding in protein structure
Authors: MAO Boryeu , CHOU Kuo‐Chen , MAGGIORA Gerald M.
Published: March 2005

A recent study has shown that topological stereoisomers exist for the polypeptide chain in disulfide‐containing proteins that are represented by non‐planar graphs. This topological stereochemistry is demonstrated in the structure of variant 3 toxin in the venom of the North American scorpion Centruroides sculpturatus Ewing and the structure of toxin II from the North African scorpion Androctonus australis Hector. In this report, we found that a similar topological analysis can be applied to the hydrogen bonding in α‐helices and β‐sheets within protein molecules, and we described the topological characteristics of chiral properties of protein secondary structure elements. Specifically, a closed right‐handed α‐helix of more than six residues long is shown formally to be non‐planar and has the L topology. Antiparallel β‐sheets are planar. Two parallel β‐strands each of at least three residues in length, however, constitute a non‐planar structural element and can have either L or D topology. The favored right‐handed crossover for parallel β‐sheets has the L form, the same as the right‐handed α‐helix. This topological description of the hydrogen bonding in secondary structures may be extended to higher levels of protein structure and may provide a conceptual framework for studying complex protein architecture in general.
NMR and molecular dynamics studies of the mKr2‘zinc finger’
Authors: CARR Mark D. , PASTORE Annalisa , GAUSEPOHL Heinrich , FRANK Rainer , ROESCH Paul
Published: March 2005

We determined the structure of a 30‐amino‐acid ‘zinc finger’ motif from the mouse Kruppel‐like gene mKr2 in solution in the absence of metal ions by two‐dimensional NMR, distance geometry and restrained molecular dynamics methods. The most prominent secondary structural feature of the peptide is a helix extending from Ser14 to Ile20. The zinc‐containing structure of the peptide was simulated by molecular dynamics calculations with restraints derived from the known geometry of the zinc ion and the ligating amino acid residues Cys6, Cys9, His22, His26. The latter structure does not deviate markedly from the set of structures determined for the zinc‐free peptide, i.e. this peptide can accommodate a Zn2+ ion without major structural rearrangements. Thus, we propose that the metal ion stabilizes the existing three‐dimensional peptide structural motif rather than introducing novel structural features.
Molecular analysis of the structural gene for yeast transaldolase
Authors: SCHAAFF Ine , HOHMANN Stefan , ZIMMERMANN Friedrich K.
Published: March 2005

We have cloned the structural gene for yeast transaldolase. Transformants carrying the TAL1 gene on a multicopy plasmid over‐produced transaldolase. A deletion mutant which was constructed using the cloned gene did not show any detectable transaldolase activity in vitro. Furthermore, both transaldolase isoenzymes which were detected in wild‐type crude extracts by immunoblotting were missing in the deletion mutants. Thus, TAL1 is the only transaldolase structural gene in yeast.

TAL1 is not an essential gene. Deletion of the transaldolase gene did not affect growth on complete media with different carbon sources or on synthetic media. However, the transaldolase‐deficient strains accumulated sedoheptulose 7‐phosphate, an intermediate of the pentose‐phosphate pathway. Mutants lacking both transaldolase and phosphoglucose isomerase grew more slowly than the single mutants. They accumulated more sedoheptulose 7‐phosphate on medium containing fructose than on glucose medium. This shows that fructose 6‐phosphate and glyceraldehyde 3‐phosphate, metabolites of glycolysis, can enter the nonoxidative part of the pentose‐phosphate pathway.
Characterization of the translational start site for IF2β, a short form of Escherichia coli initiation factor IF2
Authors: MOREL‐DEVILLE Françoise , VACHON Gilles , SACERDOT Christine , COZZONE Alain Jean , GRUNBERG‐MANAGO Marianne , CENATIEMPO Yves
Published: March 2005

The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (α and β) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigourous determination of the nature of the initiation triplet for IF2β, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2α and IF2β. Three types of experiments were carried out. First, a 77‐bp deletion was created at the beginning of the structural gene leading to premature termination of IF2α synthesis. Under these conditions, IF2β is still formed. Second, various Bal31 digests of infB containing the 77‐bp deletion were fused to lacZ. Any synthesis of a fused protein with β‐galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2β initiation site within an 18‐base region containing an in‐phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2α start sequences but containing genetic information for this 18‐base region. A hybrid protein with β‐galactosidase activity was synthesized. Moreover, its NH2‐terminal amino acid sequence coincided with that of IF2β, demonstrating that GUG, located 471 bases downstream from the IF2α external start codon, is the internal start codon for the shorter form of IF2.
Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5
Authors: HOHMANN Stefan , CEDERBERG Håkan
Published: March 2005

Recently we deleted the pyruvate decarboxylase structural gene PDC1 from the genome of the yeast Saccharomyces cerevisiae. The pdc1 deletion mutants had pyruvate decarboxylase activity due to the presence of a second structural gene [Schaaff, I., Green, J. B. A., Gozalbo, D. & Hohmann, S. (1989) Curr. Genet. 15, 75–81]. We cloned and sequenced this gene which we call PDC5. The predicted amino acid sequences of PDC1 and PDC5 are 88% identical.

Deletion of PDC5 did not cause any decrease in the specific pyruvate decarboxylase activity while pdc1 deletion mutants had 80% of the wild‐type activity. Deletion mutants lacking both PDC1 and PDC5 did not show any detectable pyruvate decarboxylase activity in vitro and were unable to ferment glucose. This indicates that PDC1 and PDC5 are the only structural genes for pyruvate decarboxylase in yeast.

The PDC5 isoenzyme showed a slightly higher Km value for its substrate pyruvate than the PDC1 product (PDC5: Km= 8 mM; PDC1: Km= 5 mM), as measured in crude extract of pdc1 and pdc5 deletion mutants, respectively.

PDC5 is only expressed in pdc1 deletion mutants. No mRNA transcribed from PDC5 could be detected in wild‐type cells. Thus, in addition to the control by glucose induction, pyruvate decarboxylase activity seems to be subject to autoregulation. Similar phenomena have been described previously for tubulin, histones and a ribosomal protein but not for metabolic enzymes.
Study of a series of analogs of salmon calcitonin in which alanine replaces leucine
Authors: EPAND Richard M. , EPAND Raquel F. , ORLOWSKI Ronald C.
Published: March 2005

Leucine residues at positions 12, 16 and 19 of salmon calcitonin were systematically replaced by alanine either one, two or three at a time. Substitution of Ala at positions 12 or 16 had the greatest effect on the structure of the peptide and on the ability of the peptide to attain structures of higher helical content in the presence of lipid. Despite the similar effects on the conformational properties, the effects on activity are markedly different for analogs with Ala substitutions at positions 12 and 16. The Ala12 derivatives are much less active while the Ala16 derivatives are slightly more active than the parent hormone. In contrast to the effects of substitutions at these positions, substitution of Ala at position 19 has relatively little effect on activity or on conformation. These results demonstrate that different regions of the calcitonin molecule have different conformational requirements for maximal activity.