bluuebunny/crossref_metadata_2025 huggingface dataset:
Radish mosaic virus VPg Characteristics and linkage with virion RNAs
Authors: Drygin Yuri F. , Sapotsky Michail V. , Bogdanov Alexey A.
Published: July 2002

Comoviruses have a bipartite RNA genome. It is suggested that a small protein, VPg, is covalently linked to both RNAs. We have found that both radish mosaic virus RNAs are linked to identical VPg molecules via a phosphodiester bond between their 5′‐terminal nucleotides and a serine residue of VPg.
Carrier‐free 8‐azidoadenosine 5′‐[γ‐32P]triphosphate
Authors: Sabbatini Gregory P. , von Holt Claus
Published: July 2002

We found 8‐azidoadenosine 5′‐diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3‐diphosphoglyceric acid to 3‐phosphoglycerate. This has allowed us to synthesize in a single‐step procedure carrier‐free 8‐azidoadenosine 5′‐[γ‐32P]triphosphate, requiring no further purification of the end product. The synthesized 8‐azidoadenosine 5′‐[γ‐32P]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue.
Demonstration of ΔpH‐ and Δψ‐induced synthesis of inorganic pyrophosphate in chromatophores from Rhodospirillum rubrum
Authors: Strid Åke , Karlsson Inga-Maj , Baltscheffsky Margareta
Published: July 2002

It is possible to obtain synthesis of PPi by artifical ion potentials in Rhodospirillum rubrum chromatophores. PPi can be formed by K+‐diffusion gradients (Δψ), H+ gradients (ΔpH) or a combination of both. In contrast, ATP can only be synthesized by imposed Δψ or Δψ+ΔpH. For ATP formation there is also a threshold value of K+ concentration below which synthesis of ATP is not possible. Such a threshold is not found for PPi formation. Both PPi and ATP syntheses are abolished by addition of FCCP or nigericin and only marginally affected by electron transport inhibitors. The synthesis of PPi can be monitored for several minutes before it ceases, while ATP production stops within 30 s. As a result the maximal yield of PPi is 200 nmol PPi/μmol BChl, while that of ATP is no more than 25 nmol ATP/μmol BChl. The initial rates of syntheses were 0.50 μmol PPi/μmol BChl per min and 2.0 μmol ATP/μmol per min, respectively. These rates are approx. 50 and 20% of the respective photophosphorylation rates under saturating illumination.
A model for the interaction of nucleic acids with transcription factor IIIA
Authors: Andersen Janet
Published: July 2002

Based on published biochemical evidence which examines the interaction of Xenopus transcription factor IIIA (TFIIIA) with 5 S RNA genes and 5 S RNA, this paper proposes that the formation of a 5 S RNA type stem‐loop structure in the DNA occurs during the binding of TFIIIA to 5 S genes.
Overexpression of mammalian phenylalanyl‐tRNA synthetase upon phenylalanine restriction
Authors: Lazard Myriam , Mirande Marc , Waller Jean-Pierre
Published: July 2002

The effect of phenylalanine restriction on the level of expression of phenylalanyl‐tRNA synthetase from cultured Chinese hamster ovary cells was investigated. By lowering the phenylalanine concentration from 200 to 2 μM, cell growth was arrested, tRNAPhe aminoacylation level was rapidly and specifically decreased and phenylalanyl‐tRNA synthetase was derepressed. The progressive 2‐fold elevation of phenylalanyl‐ tRNA synthetase level was determined by activity measurement and immunotitration. None of the other aminoacyl‐tRNA synthetases tested were significantly affected.
Transacylase activity of lactating bovine mammary fatty acid synthase
Authors: Anderson Gregory J. , Kumar Soma
Published: July 2002

An assay for the transacylation reaction catalyzed by fatty acid synthase was developed which does not require model substrates or labelled acyl‐derivatives of CoA. It involves the transfer of the acyl group from unlabelled CoA to [3H]CoA. This assay shows the occurrence of transacylation at a relatively high rate with a variety of substrates that the enzyme is able to utilize. The activity is unaffected by dissociation of the enzyme or modification by iodoacetamide or 2‐chloroacetyl‐CoA.
Proteolysis rates of a myosin heavy chain site with papain Evidence for a combined LC2‐filament‐mediated mechanism
Authors: Cardinaud R.
Published: July 2002

In striated muscle myosin, a proteolysis site at the 25–50 kDa junction, susceptible in the filament and efficiently protected by nucleotides, is similarly protected when myosin is monomeric. Kinetic studies at low ionic strength show a close relationship between LC2 cleavage or degradation rate and cleavage of the 25–50 kDa heavy chain site. The myosin‐[(T)‐LC2′] species forms normal reconstituted filaments but its 25–50 kDa site susceptibility is closer to that of monomeric myosin, thus becoming practically ionic strength‐independent. In this species the absence of the LC2 N‐terminal segment induces a significantly greater susceptibility of the papain‐sensitive site in LC1. In an LC2‐depleted myosin the 25–50 kDa site susceptibility also becomes ionic strength‐independent, however, the cleavage rates are then closer to that of filaments. Susceptibility in HMM and S1 is also much less dependent on ionic strength with rates intermediary between those of filament and monomer. These observations show that the maximum susceptibility to papain of the 25–50 kDa site requires both the integrity of the LC2 light chain and the filament structure and furthermore provide evidence that: (i) the LC2 N‐terminus interacts specifically with some part of the filament; (ii) this interaction induces a specific transconformation in a region close to the ATPase active site; (iii) there is an interrelationship between LC1 and LC2 light chain N‐terminal extremities, at least in the filament structure.
Neurohypophyseal peptide inhibition of adenylate cyclase activity in fish gills The effect of environmental salinity
Authors: Guibbolini Marielle E. , Lahlou Brahim
Published: July 2002

Adenylate cyclase activity was measured in plasma membranes prepared from gill epithelium of the rainbow trout (Salmo gairdneri) adapted to a large range of salinity (deionized water to seawater). Fish neurohypophyseal peptides (arginine‐vasotocin and isotocin) elicited a dose‐dependent inhibition (maximum for 10−12−10−10 M which corresponds to physiological blood concentrations) of both basal and 10−9 M glucagon stimulated enzyme activity. While basal activity was inhibited by up to 40% only in high salt media, glucagon‐stimulated activity was lowered by 60–100% for the same concentrations, in both freshwater and seawater.
Methylation of the enhancer region of avian sarcoma virus long terminal repeat suppresses transcription
Authors: Guntaka Ramareddy V. , Gowda Siddareme , Wagner Herbert , Simon Dietrich
Published: July 2002

The effect of methylation of an enhancer on transcription was studied. A 245 bp enhancer‐containing a fragment of the LTR of the avian sarcoma virus was methylated in vitro and ligated back into a vector which lacked the upstream enhancer sequence. The transient expression in QT6 cells indicated that methylation of the enhancer‐containing sequence severely reduced the extent of transcription.
Evidence for a phosphorylation‐induced conformational change in phospholamban from the effects of three proteases
Authors: Huggins John P. , England Paul J.
Published: July 2002

The effect of the proteases trypsin, thermolysin and papain on the cardiac membrane protein phospholamban was examined before or after phosphorylating the protein with the catalytic subunit of cyclic AMP‐dependent protein kinase. The sensitivity of phospholamban to digestion by trypsin and thermolysin was greatly reduced by phosphorylation, suggesting that phospholamban undergoes a conformational change upon phosphorylation. It is suggested that this change in conformation is the mechanism by which phospholamban phosphorylation relieves its inhibition of the sarcoplasmic reticulum Ca2+‐ATPase pump.
Platelet‐derived growth factor Phorbol ester induces the expression of the B‐chain but not of the A‐chain in HEL cells
Authors: Weich H.A. , Herbst D. , Schairer H.U. , Hoppe J.
Published: July 2002

It was shown previously [(1984) EMBO J. 3, 453‐459] that after treatment of the human erythroleukemia cell line HEL with phorbol ester and dimethyl sulfoxide there was a marked increase in the amounts of megakaryotic markers, especially of platelet α‐granule proteins and platelet glycoproteins. In order to investigate this differentiation process further we have studied the expression of the mRNA encoding PDGF‐A and PDGF‐B (c‐sis). Upon addition of the phorbol ester to the culture medium the expression of the c‐sis transcript was enhanced about 7‐fold over a period of 4 days. With dimethyl sulfoxide there was no significant stimulation of the expression. Addition of cycloheximide to HEL cells treated for a short period with phorbol ester superinduced the expression of the c‐sis gene. The HEL cells did not express the A‐chain mRNA even in the presence of phorbol ester or dimethyl sulfoxide. This leads us to propose that synthesis of the PDGF‐A chain and PDGF‐B chain is differentially regulated in the megakaryocytic‐like HEL cell line.
A 5 S rRNA‐like secondary structure in the 7 SL RNA may define a ribosomal binding site of the signal recognition particle
Authors: Boehm Siegfried
Published: July 2002

A new secondary structure model for parts of the 7 SL RNA is proposed which indicates for a stretch of at least 40 bases a strong structural homology to the ribosomal protein L5 binding site of eukaryotic 5 S rRNA. It is suggested that the 5 S rRNA‐like structural part of 7 SL RNA mediates binding of the signal recognition particle near to the peptidyl transferase center of the ribosome.
Primary structure of rat liver serine dehydratase deduced from the cDNA sequence
Authors: Noda Chiseko , Ito Keiko , Nakamura Toshikazu , Ichihara Akira
Published: July 2002

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer‐extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3′‐ and 5′‐sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly‐Ser‐Phe‐Lys‐Ile‐Arg‐Gly, which is the pyridoxal‐binding site in the yeast and E. coli threonine dehydratases, was found as a highly conserved sequence.
Selective inhibition of oat glutathione‐S‐transferase activity by tetrapyrroles
Authors: Singh Bal Ram , Shaw Robert W.
Published: July 2002

Inhibition of glutathione‐S‐transferase activity by various tetrapyrroles (bilirubin, biliverdin, chlorophyllin and hemin) has been studied. Addition of 77 μM bilirubin or biliverdin inhibited glutathione‐S‐transferase activity only by 15–19% whereas 77μM chlorophyllin or hemin inhibited the enzyme activity by 83–86%. The data suggest that oat glutathione‐S‐transferases can distinguish between open and closed tetrapyrroles. Possible physiological significance of the observed results is discussed.
Location of divalent ion sites in acyl carrier protein using relaxation perturbed 2D NMR
Authors: Frederick Anne F. , Kay Lewis E. , Prestegard James H.
Published: July 2002

The T
1‐accordion COSY experiment has been applied to acyl carrier protein (ACP) to locate the divalent ion binding sites in the protein using the paramagnetic ion, Mn2+, as a substitute for Ca2+. Replacement with Mn2+ leads to an enhancement of proton spin‐lattice (T
1) relaxation rates. These enhancements have a l/r
6, distance dependence that makes them extremely useful in structural analyses. Ion‐proton distances ranging from 3.0 to 9.0 Å have been obtained from this experiment and subsequently used as constraints in the molecular mechanics module of AMBER to refine a protein structure.
Purification of fusion proteins expressed by pEX3 and a truncated pEX3 derivative
Authors: Kocken Clemens H.M. , Scheer J.M.José , Welling Gjalt W. , Welling-Wester Sytske
Published: July 2002

A derivative of the pEX3 expression vector was constructed that codes for the first 407 amino acids of the 1051 amino acids of the pEX3 fusion protein. The amount of truncated fusion protein (40 mg/g cells), obtained by expression in E. coli, was similar to that produced by the original pEX3 vector. The truncated fusion protein was purified more easily from E. coli contaminants than the original fusion protein by washing with 2 M urea and 0.5% Triton X‐100.
Structural basis for autogenous regulation of Xenopus laevis ribosomal protein L1 synthesis at the splicing level
Authors: Gultyaev A.P. , Shestopalov B.V.
Published: July 2002

It is known that the injection of the Xenopus laevis ribosomal protein L1 gene into oocytes causes the accumulation of immature L1 transcripts due to a specific block of splicing of the second and third introns. In this paper the secondary structures of these introns in pre‐mRNA have been constructed. It has been shown that they share homology with 28 S rRNA. The putative RNA‐binding segment of L1 has also been predicted. These results are interpreted as the structural basis for autogenous regulation of X. laevis ribosomal protein L1 synthesis at the splicing level.
The binary complex of pig plasma gelsolin with Mg2+‐G‐actin in ATP and ADP
Authors: Harris Harriet E.
Published: July 2002

Pig plasma gelsolin combined with Mg‐G‐actin at < 10−8 M Cal2+ to yield a binary complex. Complexes formed from G‐actin with bound ATP or ADP. They contained approx. 1 mol of non‐exchangeable nucleotide per mol of actin. ATP hydrolysis was not coupled to binary complex formation, but ATP in the complex hydrolysed very slowly. The nucleotide in the binary complex behaved like one of the two nucleotide molecules in the ternary complex (two actin monomers to one gelsolin), but the actin‐gelsolin interaction was weaker in the binary complex.
An initiator protein for plasmid R6K DNA replication Mutations affecting the copy‐number control
Authors: Inuzuka Manabu , Wada Yukie
Published: July 2002

Two kinds of mutations affecting the copy‐number control of plasmid R6K were isolated and identified in an initiator π protein by DNA sequencing. Firstly, a temperature‐sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305‐amino‐acid π protein. Secondly, a high‐copy‐number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild‐type allele in the copy control.
Nicotinic acetylcholine receptor: A structural model for α‐subunit peptide 188–201, the putative binding site for cholinergic agents
Authors: Gotti C. , Frigerio F. , Bolognesi M. , Longhi R. , Racchetti G. , Clementi F.
Published: July 2002

A peptide corresponding to amino acid sequence 188–201 of the α‐subunit of Torpedo AChR binds α‐Bgtx. The S‐S bridge between Cys 192 and 193 is essential for the binding as Tyr in position 189. The same sequence 188–201 corresponding to human AChR, which instead of Tyr has a Thr in position 189, binds α‐Bgtx with a much lower efficiency. Monoclonal antibodies raised against Torpedo peptide 188–201 recognize Torpedo AChR and antibodies against Torpedo AChR recognize peptide 188–201 indicating that the synthetic peptide and the corresponding sequence in the native molecule share some immunological epitopes. With computer graphics and energy refinement a molecular model of this peptide has been elaborated.
Limited proteolysis of 3‐phosphoglycerate kinase without loss of enzymic activity
Authors: Jiang Shu-Xian , Vas Mária
Published: July 2002

During tryptic digestion of pig muscle 3‐phosphoglycerate kinase in the presence of 3‐phosphoglycerate both the decrease of enzymic activity and the release of trichloroacetic acid‐soluble peptides occur after a pronounced lag period. During this lag phase the native enzyme molecule is split into two fragments with molecular masses of about 30 and 18 kDa, as detected by SDS‐PAGE. Under non‐denaturing conditions, however, these fragments are held together by non‐cova‐lent forces and constitute an active, nicked enzyme molecule. In the absence of substrates or in the presence of MgATP the kinetics of tryptic digestion is apparently a single first order reaction leading to the formation of peptides with molecular masses of less than 10 kDa.
Diol lipids are activators of protein kinase C
Authors: Severin Sergey E. , Tovmasyan Eranui K. , Shvets Vitaly I. , Molotkovsky Julian G. , Bergelson Lev D.
Published: July 2002

Diol lipids (dioleoyl‐ and dioctanoylethylene glycol) at relatively low concentrations (∼ 10 μM) were found to activate significantly protein kinase C in the presence of phosphatidylserine or phosphatidylinositol. Since diol lipids are widespread minor lipid constituents of many cells [(1974) Chem. Ind., 597–604], it has been suggested that they may be involved in the maintaining of basal protein kinase C activity in the absence of external stimuli.
Variable RNA polymerase populations in the life cycle of Trypanosoma brucei
Authors: Tittawella Ivor
Published: July 2002

DNA‐dependent RNA polymerase from blood forms and culture forms of the parasitic protozoan Trypanosoma brucei was resolved into multiple peaks of activity by DEAE‐Sephadex chromatography. The enzyme from the two forms was found to be different by several criteria, suggesting that it is subject to developmental control.
The amino acid substitution in albumin Roma: 321 Glu → Lys
Authors: Galliano M. , Minchiotti L. , Iadarola P. , Ferri G. , Zapponi M.C. , Castellani A.A.
Published: July 2002

Albumin Roma is an electrophoretically slow moving genetic variant of human serum albumin found in 22 unrelated families. The protein was isolated from the serum of a healthy, heterozygous subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to fragment CNBr IV (residues 299–329). This fragment was isolated on a preparative scale by RP‐HPLC and subjected to tryptic digestion. Sequential analysis of two abnormal tryptic peptides, purified by RP‐HPLC, revealed that the variant arises from the substitution of glutamic acid 321 by lysine. This amino acid replacement, probably resulting from a point mutation in the structural gene, causes a change in the net charge of +2 units which is in keeping with the decreased electrophoretic mobility of the native protein.
Three‐dimensional location of ribosomal protein BL2 from Bacillus stearothermophilus, a key component of the peptidyl transferase center
Authors: Hackl W. , Stöffler-Meilicke M. , Stöffler G.
Published: July 2002

Protein BL2 from Bacillus stearothermophilus has been localized by immunoelectron microscopy on the interface side of the 50 S subunit, beneath the angle formed between the central protuberance and the L1 protuberance. The immunoelectron microscopic data suggest that the interface region of the 50 S particle is not as flat as most of the proposed three‐dimensional models suggest, but instead there is a significant concavity. Since several studies demonstrated that BL2 is implicated in peptidyl transferase activity or at least located close to the peptidyl transferase center, the location of protein BL2 also provides information as to the location of this important functional domain.
New fluoroketones as human renin inhibitors
Authors: Tarnus Céline , Jung Michel J. , Rémy Jean-Marc , Baltzer Sylvie , Schirlin Daniel G.
Published: July 2002

Renin inhibition has been evaluated for a new class of fluorinated ketones, true analogues of peptides that have been retroinverted at the C‐terminal position. The readily formed hydrate of the ketone is proposed to mimic the tetrahedral intermediate that occurs during the enzyme‐catalyzed hydrolysis of amide linkage. From this series of compounds it appears that the number of reverted amide bonds is crucial in terms of activity. Furthermore, a shortening of the C‐terminal part of our peptide analogues and the replacement of the leucine residue in P1 by a cyclohexylalanine leads to the tripeptide analogue 12 a potent renin inhibitor (IC50 = 3.5 × 10−9 M).
Forskolin decreases sensitivity of brain adenylate cyclase to inhibition by 2′,5′‐dideoxyadenosine
Authors: Stübner Dorothee , Johnson Roger A.
Published: July 2002

The effects of forskolin on the sensitivity of adenylate cyclase to ‘P’‐site‐mediated inhibition were studied. Stimulation of crude and purified preparations of adenylate cyclase by forskolin led to decreased sensitivity to inhibition by 2′,5′dideoxyadenosine with enzyme from rat and bovine brain. This is in contrast with the enhancement of P‐site sensitivity induced by calmodulin, divalent cations, and stable GTP analogs and is in contrast with behavior seen with enzyme from liver and S49 cyc− membranes. The effect of forskolin on P‐site sensitivity of the brain adenylate cyclase was not dependent on the presence of G‐proteins or calmodulin. It was not the consequence of proteolysis nor was it due to an obvious artifact in the assay procedures. This distinct behavior of the brain enzyme is most likely due to a structural difference in the catalytic subunit.
Probing the topography of the intramembrane part of Na+,K+‐ATPase by photolabelling with 3‐(trifluoromethyl)‐3_(m[125I]iodophenyl)diazirine Analysis of the hydrophobic domain of the β‐subunit
Authors: Chertova E.N. , Lutsenko S.V. , Levina N.B. , Modyanov N.N.
Published: July 2002

The mutual disposition of the α‐helical intramembrane rods in Na+,K+‐ATPase subunits was studied by photolabelling of the membrane‐bound enzyme with 3‐(trifluoromethyl)‐3‐(m‐[125I]iodophenyl)diazirine ([125I]TID). Under the chosen conditions for modification, the ratio of label incorporated into the subunits was found to be α/β = 2.2, demonstrating the peripheral location of the β‐subunit in the oligomeric membrane complex. The [125I]TID‐labelled β‐subunit was subjected to tryptic hydrolysis and the modified fragment (Thr27‐Arg71) was isolated. The labelled amino acid residues are located predominantly on one side of the helix, which is helpful in unravelling the spatial orientation of the β‐subunit relative to the α‐subunit.
Remarks on the supramolecular organization of the glycolytic system in vivo
Authors: Batke J.
Published: July 2002

The great latent catalytic capacity, manifested at the extremely high intracellular concentrations and in large apparent k
cat/K
m values, of the glycolytic enzymes on the one hand and their tendency in experiments in vitro to form functionally‐specific flux‐enhancing (channeling) complexes on the other, is considered and discussed as an apparent discrepancy. A random association of glycolytic enzymes in vivo is probable.
Lumen‐side topography of the α‐subunit of the chloroplast cytochrome b‐559
Authors: Tae G.-S. , Cramer W.A.
Published: July 2002

Removal of the extrinsic 33 kDa polypeptide increased the accessibility to trypsin of a COOH‐terminal tridecapeptide epitope of the α subunit of cytochrome b‐559 (psbE. gene product). The sensitivity of the cytochrome epitope to trypsin was not measurably affected by removal of the 16 and 23 kDa extrinsic polypeptides, nor increased by removal of the OEC manganese along with the 33 kDa protein. While protecting α‐cytochrome b‐559 against trypsin, the 33 kDa protein is also proteolyzed, suggesting the possibility of an additional protein component involved in the shielding of the cytochrome. Shielding of the COOH‐terminal epitope of α‐cytochrome b‐559 by the OEC 33 kDa protein implies that these COOH‐terminal chains of the cytochrome are part of a protein network in the lumen space near the photosystem II reaction center. This network may contain residues that are involved in the binding of essential OEC metal ions.
Expression of liver‐specific genes coding for plasma proteins in protein deficiency
Authors: Perozzi Giuditta , Mengheri Elena , Faraonio Raffaella , Gaetani Sancia
Published: July 2002

Protein deficiency leads to a decreased concentration of plasma proteins, although it is not clear whether this response is caused by alterations in gene transcription or in post‐transcriptional events. The aim of this study was to investigate the expression of some liver‐specific genes coding for plasma proteins in rats kept on a protein‐free diet for 30 days. Cloned cDNA probes for the albumin, transthyretin, retinol‐binding protein and prothrombin genes were used in Northern hybridizations to total liver RNA to compare their transcript levels in protein‐deficient and control animals. Liver polysomes were also isolated and fractionated from the two groups of animals to examine the possible effects of protein deficiency on translation of the mRNAs. The results indicate that the albumin and transthyretin mRNAs are present in lower amounts in protein deficiency. The distribution profile along sucrose gradients shows that all mRNAs are undergoing translation, but in protein‐deficient animals a small but consistent fraction of each mRNA is also present in the non‐polysomal, low molecular weight fractions.
Multiple GF‐1 binding sites flank the erythroid specific transcription unit of the human carbonic anhydrase I gene
Authors: Brady Hugh J.M. , Sowden Jane C. , Edwards Mina , Lowe Nicholas , Butterworth Peter H.W.
Published: July 2002

Six potential GF‐1 sites which bind an erythroid factor are present in the 5' and 3' regions flanking the erythroid‐speciflc transcription unit of the human carbonic anhydrase 1 (HCAI) gene. When two of these sites are placed upstream of a minimal eukaryotic promoter they confer upregulated expression in erythroid over non‐erythroid cells. The presence of the erythroid factor in TPA‐treated HEL cells in which the level of HCAI transcript has greatly decreased and in non‐HCAI‐expressing K562 cells suggests that in these cases the presence of the factor is not sufficient for HCAI expression.
Phosphorus‐containing inhibitors of aspartate transcarbamoylase from Escherichia coli
Authors: Laing Naomi , Chan William W.-C. , Hutchinsoi David W. , Öberg Bo
Published: July 2002

A tetrahedral intermediate is the prominent feature of the generally accepted mechanism for aspartate transcarbamoylase. We have synthesized N‐pyrophosphoryl‐L‐aspartate as a charged analogue of the postulated intermediate. Surprisingly, its affinity for the enzyme from Escherichia coli was substantially lower than that of the previously known inhibitor phosphonoacetyl‐L‐aspartate which contained a trigonal carbonyl group. Similar results were obtained with the corresponding mercaptosuccinate derivatives. We also tested a number of new pyrophosphate analogues as inhibitors. Our results cast doubt on some aspects of the current model for the mechanism of this enzyme.
Construction of expression plasmids for the fusion protein of Sendai virus, and their expression in E. coli cells and eucaryotic cells
Authors: Taira Hideharu , Ranin Lazar , Iwasaki Kentaro
Published: July 2002

To examine the properties and the role of the fusion protein (F) of Sendai virus at the molecular level, a plasmid, pUC‐F, was constructed by inserting cDNA for the F protein into a pUC vector. Upon induction of E. coli cells transformed with pUC‐F, a new protein was obtained, which was identified as Fo on Western blot analysis. The cDNA fragment for the F gene was excised from pUC‐F and inserted into an eucaryotic expression vector, pSVL, to yield pSVL‐F. COS‐1 cells transfected with pSVL‐F gave a band on SDS‐gel electrophoresis which corresponded to the size of the Fo protein.
Prenyltransferase compartmentation in cells of Vitis vinifera cultivated in vitro
Authors: Feron G. , Clastre M. , Ambid C.
Published: July 2002

Two prenyltransferases were located in cell cultures of Vitis vinifera. A geranyl pyrophosphate synthase (EC 2.5.1.1) was associated with plastid‐like membranes whereas a farnesyl pyrophosphate synthase (EC 2.5.1.10) was found to be soluble.
Competitive interaction of three peroxidizing herbicides with the binding of [3H]acifluorfen to corn etioplast membranes
Authors: Varsano Rina , Matringe Michel , Magnin Noël , Mornet René , Scalla René
Published: July 2002

The specific binding of the herbicide acifluorfen 5‐[2‐chloro‐4‐(trifluoromethyl)phenoxy]‐2‐nitrobenzoic acid to corn etioplast membranes is competitively inhibited by protoporphyrinogen IX, the substrate of protoporphyrinogen oxidase. Three other peroxidizing molecules, oxadiazon [5‐ter‐butyl‐3‐(2,4‐dichloro‐5‐isopropoxyphenyl)‐1,3,4‐oxadiazol‐2‐one], LS 82556 [(S)3‐N‐(methylbenzyl)carbamoyl‐5‐propionyl‐2,6‐lutidine], and M&B 39279 [5‐amino‐4‐cyano‐1‐(2,6‐dichloro‐4‐trifluoromethylphenyl)pyrazol], also compete with acifluorfen for its binding site. The four herbicides thus bind to the same site, or to closely located sites, on the enzyme protoporphyrinogen oxidase.
The primary structure of the γ‐subunit of the ATPase from Synechocystis 6803
Authors: Werner Sabine , Schumann Jürgen , Strotmann Heinrich
Published: July 2002

The nucleotide sequence of the gene coding for the FoF1‐ATPase γ‐subunit (atpC) from the transformable cyanobacterium Synechocystis 6083 has been determined. The deduced translation product consists of 314 amino acid residues and is highly homologous (72% identical residues) to the sequences of other cyanobacterial γ‐subunits. The Synechocystis 6803 sequence is also homologous to the chloroplast γ‐sequence. Like in the other cyanobacterial subunits, only the first of the 3 cysteine residues, which are involved in energy‐linked functions of the γ‐subunit in spinach chloroplasts, is conserved in Synechocystis 6803.
The electric unit size of thylakoid membranes
Authors: Schönknecht G. , Althoff G. , Junge W.
Published: July 2002

The size of the function unit of electrical events in thylakoid membranes was estimated by the minimum amount of gramicidin needed to discharge the flash light generated electrical potential difference. Early flash spectroscopic measurements have indicated that a single gramicidin dimer operates on an electrical function unit containing at least 2 × 105 chlorophyll molecules [1]. In this study we present gramicidin titrations with more intact thylakoid preparations which revealed a more than hundred‐fold greater lower limit for the electric unit size, namely 5 × 107 chlorophyll molecules. It is conceivable that the whole complicated thylakoid structure inside a chloroplast constitutes a single electric unit. It comprises more than 2 × 108 chlorophyll molecules in an area of more than 400 μm2.
Studies on the primary sequence requirements for PKC‐α, ‐β1 and ‐γ peptide substrates
Authors: Marais Richard M. , Nguyen Oanh , Woodgett James R. , Parker Peter J.
Published: July 2002

The substrate specificity of purified PKC‐α, ‐β and ‐γ has been investigated. A series of synthetic peptides based upon the sequence surrounding serine‐7 in glycogen synthase were generated and used to determine the basic residue requirements of these PKC isotypes. While PKC‐α and ‐β are indistinguishable in their phosphorylation of these peptides, PKC‐γ shows a distinct specificity profile for these synthetic substrates.
Calcium‐permeable channels activated via guanine nucleotide‐dependent mechanism in human carcinoma cells
Authors: Mozhayeva Galina N. , Naumov Alexander P. , Kuryshev Yury A.
Published: July 2002

Patch clamp experiments on human carcinoma A431 cells have revealed two types of Ca2+‐permeable channels, the activity of which can be increased by the application of non‐hydrolyzable analogues of GTP to the intracellular side of the membrane. With 105 mM Ca2+ in recording pipette at 30–33°C their unitary conductances (in pS) are 1.3 (SG‐channels) and 2.4 (G‐channels). G‐ and, possibly, SG‐channels are activated from the extracellular side of the membrane with epidermal growth factor (EGF). The data are consistent with the hypothesis that both channels are activated via guanine nucleotide binding (G) proteins.
Does the Kunitz domain from the Alzheimer's amyloid β protein precursor inhibit a kallikrein responsible for post‐translational processing of nerve growth factor precursor?
Authors: Castro Maria , Marks Cara Berman , Nilsson Björn , Anderson Stephen
Published: July 2002

Alternative splicing of the Alzheimer's amyloid β protein precursor (ABPP) message leads to the production of several variants of this precursor polypeptide. Two of these variants contain a domain that is highly homologous to members of the Kunitz class of protease inhibitors. In order to initiate a study of the physiological role of this domain, we have produced active ABPP Kunitz inhibitor by constructing and expressing a synthetic gene in E. coli. Nerve growth factor (NGF) deficiency has been suggested as a possible cause of the neural degeneration characteristic of Alzheimer's disease, and trypsin and γ‐NGF are the two enzymes that have been shown to be capable of processing β‐NGF precursor to active, mature β‐NGF in vitro, therefore the specificity of purified recombinant ABPP Kunitz inhibitor was analyzed with respect to these two proteases. Binding of isolated ABPP Kunitz domain both to trypsin (K
i,app < 10 nM and to γ‐NGF (K
i,app = 300 nM) was observed. This difference in binding to the two proteases correlates with the approximately 20‐fold higher rate observed for in vitro processing of the β‐NGF precursor by trypsin compared to processing by γ‐NGF, indicating that perhaps the inhibitor mimics the interaction of the β‐NGF precursor with proteases. The kallikrein actually responsible for β‐NGF precursor processing in vivo is unknown, but these results suggest that it is capable of being significantly inhibited by exposure to the ABPP Kunitz domain.
Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes
Authors: Klibanov Alexander L. , Maruyama Kazuo , Torchilin Vladimir P. , Huang Leaf
Published: July 2002

Incorporation of dioleoyl N‐(monomethoxy polyethyleneglycol succinyl)phosphotidylethanolamine (PEG‐PE) into large unilamellar liposomes composed of egg posphatidylcholine:cholesterol (1:1) does not significantly increase the content leakage when the liposomes are exposed to 90% human serum at 37°C, yet the liposomes show a significant increase in the blood circulation half‐life (t

= 5 h) as compared to those without PEG‐PE(t

<30 min). The PEG‐PE's activity to prolong the circulation time of liposomes is greater than that of the ganglioside GM1, awell‐described glycolipid with this activity. Another amphipathic PEG derivative, PEG stearate, also prolongs the liposome circulation time, although its activity is less than that ofGM1. Amphipathic PEGs may be useful for the sustained release and the targeted drug delivery by liposomes.
Production and purification of a granular‐starch‐binding domain of glucoamylase 1 from Aspergillus niger
Authors: Belshaw Nigel J. , Williamson Gary
Published: July 2002

A domain of glucoamylase 1 from Aspergillus niger which binds to granular starch was produced by proteolytic digestion and purified to apparent homogeneity by extraction with corn starch followed by anion‐exchange chromatography and gel filtration. The peptide has a molecular weight of 25100, contains approximately 38% carbohydrate () and corresponds to residues 471–616 at the C‐terminus of glucoamylase 1. The peptide bound to granular corn starch maximally at 1.08 starch. It inhibited the hydrolysis of granular starch by glucoamylase 1 but had no effect on the hydrolysis of starch in solution.
Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases
Authors: Doctor B.P. , Chapman T.C. , Christner C.E. , Deal C.D. , De La Hoz D.M. , Gentry M.K. , Ogert R.A. , Rush R.S. , Smyth K.K. , Wolfe A.D.
Published: July 2002

The complete amino acid sequence of a mammalian acetylcholinesterase from fetal bovine serum (FBS AChE) is presented. This enzyme has a high degree of sequence identity with other cholinesterases, liver carboxyesterases, esterase‐6, lysophospholipase, and thyroglobulin. The locations of 191 amino acids in 10 regions of the FBS enzyme were compared with corresponding sequences of Torpedo, human, and Drosophila AChEs and human serum butyrylcholinesterase (BChE). In one region there is a marked difference in both the number of amino acids and their sequence between mammalian AChE and other AChEs and the human serum BChE. The amino acid sequence of FBS AChE showed overall homologies of 90% with human AChE, 60% with T. californica AChE, 50% with human serum BChE, and 39% with Drosophila AChE in these regions.
Identification of sulfurtransferase enzymes in Azotobacter vinelandii
Authors: Pagani S. , Franchi E. , Colnaghi R. , Kennedy C.
Published: July 2002

Rhodanese and 3‐mercaptopyruvate sulphurtransferase have been identified in A. vinelandii. Two distinct active fractions of the two sulphurtransferases were obtained after FPLC ion‐exchange chromatography of material partially purified from crude extracts. Rhodanese has been purified to homogeneity, and it consists of one polypeptide chain of M
r ca 25 000. A partial purification of 3‐mercaptopyruvate sulphurtransferase was obtained.
Cloning sequencing of Lol pI, the major allergenic protein of rye‐grass pollen
Authors: Griffith Irwin J. , Smith Penelope M. , Pollock Joanne , Theerakulpisut Piyada , Avjioglu Asil , Davies Sean , Hough Terryn , Singh Mohan B. , Simpson Richard J. , Ward Larry D. , Knox R.Bruce
Published: July 2002

We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI‐specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen‐allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye‐grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure—function relationship of allergens.
Novel epoxysuccinyl peptides Selective inhibitors of cathepsin B, in vitro
Authors: Murata Mitsuo , Miyashita Satsuki , Yokoo Chihiro , Tamai Musaharu , Hanada Kazunori , Hatayama Katsuo , Towatari Takae , Nikawa Takeshi , Katunuma Nobuhiko
Published: July 2002

A series of new epoxysuccinyl peptides were designed and synthesized to develop a specific inhibitor of cathepsin B. Of these compounds, N‐(L‐3‐trans‐ethoxycarbonyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline (compound CA‐030) and N‐(L‐3‐trans‐propylcarbamoyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline (compound CA‐074) were the most potent and specific inhibitors of cathepsin B in vitro. The carboxyl group of proline and the ethyl ester group or n‐propylamide group in the oxirane ring were necessary, the ethyl ester group or the n‐propylamide group being particularly effective for distinguishing cathepsin B from other cysteine proteinases such as cathepsins L and H, and calpains.
Noradrenergic inhibition and voltage‐dependent facilitation of ω‐conotoxin‐sensitive Ca channels in insulin‐secreting RINm5F cells
Authors: Aicardi G. , Pollo A. , Sher E. , Carbone E.
Published: July 2002

We found that, besides dihydropyridine‐sensitive Ca channels, insulin‐secreting RINm5F cells also contain a minority (15–25%) of ω‐conotoxin (ω‐CgTx)‐sensitive channels that show a high‐affinity binding to [125I]ω‐CgTx (K
d51 pM). Noradrenaline (NA, 10μM) slows down Ca‐channel activation in these cells and produces a sizeable reduction of Ca currents that is relieved by strong pre‐conditioning depolarizations (facilitation). The action of NA is mimicked by intracellular application of GTP‐γ‐S and is prevented by pertussis toxin (PTX) or by cell pre‐incubation with ω‐CgTx. This suggests specific noradrenergic inhibition of ω‐CgTx‐sensitive Ca channels that is modulated by membrane potentials and PTX‐sensitive‐G‐protein activation.
Activation‐dependent changes in microenvironment of spinach ribulose 1,5‐bisphosphate carboxylase/oxygenase
Authors: Purohit Ulka S. , Verma Naresh C. , Bhagwat Anil S.
Published: July 2002

The spinach ribulose 1,5‐bisphosphate carboxylase/oxygenase was labelled with o‐phthalaldehyde, which forms a stable fluorescent isoindole adduct at the active site. The fluorescence behaviour of the labelled enzyme after activation to different levels by Mg2+ was compared with that of a synthetic isoindole adduct of o‐phthalaldehyde, namely 1‐(hydroxyethylthio)‐2‐β hydroxyethylisoindole in solvents of different pH and polarity. The results suggest that the microenvironment at the catalytically incompetent active site of the unactivated Rubisco is highly acidic (pH < 2) in nature. The activation by Mg2+ results in the conformational change such that the effective pH at the active site increases to > 8. The polarity of the active site of the activated enzyme was found to be similar to that of a mixture of hexane and toluene.
Beef‐heart mitochondrial F1‐ATPase can use endogenous bound phosphate to synthesize ATP in dimethyl sulfoxide
Authors: Beharry Seelochan , Bragg Philip D.
Published: July 2002

Beer‐heart mitochondrial F1‐ATPase contained 5 mol of inorganic phosphate bound per mol of F1, following pretreatment with ATP. A portion of the phosphate, bound most likely at a catalytic site, reacted in dimethylsulfoxide with endogenous adenine nucleotide to form ATP.
Cloning and sequence analysis of the phenylalanyl‐tRNA synthetase genes (pheST) from Thermus thermophilus
Authors: Keller Barbara , Kast Peter , Hennecke Hauke
Published: July 2002

While crystals suitable for X‐ray diffraction analyses are available of phenylalanyl‐tRNA synthetase (PheRS) from the thermophilic bacterium Thermus thermophilus, neither the primary structure of its constituent α and β subunits nor the nucleotide sequence of the corresponding pheS and pheT genes were known. Using specific oligonucleotides of conserved pheS regions that were adapted to the T. thermophilus codon usage, we identified, cloned and subsequently sequenced the pheST genes of this bacterium. The sequences reported here will greatly aid in the three‐dimensional structure determination of T. thermophilus PheRS, a heterotetrameric (α,β2) class II aminoacyl‐tRNA synthetase.
Stable expression of high affinity NK1 (substance P) and NK2(neurokinin A) receptors but low affinity NK3 (neurokinin B) receptors in transfected CHO cells
Authors: Gether Ulrik , Marray Taisser , Schwartz Thue W. , Johansen Teit E.
Published: July 2002

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabeled substance P and neurokinin A (substance K) to CHO clones expressing the NK1 and NK2 receptors, respectively, were saturatable and of high affinity (K
d1=0.17 nM (NK1); 3.4 nM (NK2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK1 and NK2 receptors. In contrast, the binding of eledoisin to the NK3 receptor expressed in the transfected CHO cells was of low affinity (IC50=240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS‐7 cells (IC50=8 nM). However, in both cases the receptor exhibited the specificity of a classical NK3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.
Evidence of a tertiary interaction functional in group I 3′‐splicing
Authors: Fernández Ariel , Belinky Alejandro
Published: July 2002

It has been recently shown that the schedule of 3′‐splicing events for yeast mitochondrial group I introns requires that conserved helix P10 materializes only after 5′‐cleavage has taken place. A scenario compatible with experimental findings has been proposed [(1992) FEBS Lett. 297. 201–204; (1990) Proc. Natl. Acad. Sci. USA 87, 8192–8196] where the formation of P10 is postponed by competition for standard basepairing with an extended P1 interaction engaging the same portion of the internal guide sequence (IGS) which is subsequently involved in P10. Although the formation of P10 in the fifth intron of yeast apocytochrome b gene (YCOB5) has been confirmed by site‐directed mutagenesis, we cannot be confident that this interaction is merely stabilized by Watson‐Crick base‐pairinq, involving the 3′‐exon and the IGS, especially given that its formation requires closure of an intron loop of 159 unpaired bases. Thus, our aim is to establish the participation of the 5′‐extremity of the intron in the formation of P10. By deoxyribose substitution at positions 1 and 2 of the 5′‐extremity of the intron, we are able to confirm the existence of tertiary interactions stabilizing the 3′‐splicing site. We show that selective deoxyribose substitution renders the intron inefficient for 3′‐splicing when compared with wild type levels.
Unexpected intron location in non‐vertebrate globin genes
Authors: Moens Luc , Vanfleteren Jacques , De Baere Ivo , Jellie Anna M. , Tate Warren , Trotman Clive N.A.
Published: July 2002

The Caenorhabditis elegans and Artemia T4 globin sequences are highly homologous with other invertebrate globins. The intron/exon patterns of their genes display a single intron in the E and G helices respectively, Precoding introns in multirepeat globins are inserted in homologous positions. Comparison of the intron/exon patterns in the known globin gene sequences demonstrates that they are more diverse than first expected but nevertheless can be derived from an ancestral pattern having 3 introns and 4 exons.
The metastatic potential of rat prostate tumor variant R3327‐MatLyLu is correlated with an increased activity of N‐acetylglucosaminyl transferase III and V
Authors: Easton Ellen W. , Blokland Irene , Geldof Albert A. , Rao B.Ramanath , van den Eijnden Dirk H.
Published: July 2002

Enzyme activities of N‐acetylglucosaminyltransferase (GlcNAc‐Tase) I–V involved in N‐linked complex‐type carbohydrate synthesis were determined in a non‐metastatic hormone‐dependent rat prostate tumor (R3327‐H) and a related, hormone‐independent variant metastasizing to lymph nodes and lungs (R3327‐MatLyLu). In the metastasizing variant a significantly increased activity of both GlcNAc‐Tase III and GlcNAc‐Tase V was observed, whereas the activities of GlcNAc‐Tase I and II were essentially unchanged. The increase in activity of GlcNAc‐Tase III is particularly noteworthy since it indicates that elevated expression of this enzyme cannot be considered as an exclusive marker of hepatic malignancy.
Selection of mouse cells with amplified metallothionein genes retaining their glucocorticoid inducibility
Authors: Thibodeau J. , De Souza C. , Smorawinska M. , Thirion J.-P.
Published: July 2002

Two new mouse cell mutants, resistant to either 80 or 100 mM CdCl2, were isolated to study the regulation of transcription by the glucocorticoid hormones. Their metallothionein mt‐1+ and mt‐2+ genes were amplified coordinately to a maximum of 30 copies per cell. By Southern blot analysis, no gross rearrangement was detectable near the mt
+ loci. Contrary to other mutants previously isolated, the metallothionein‐specific mRNAs of these mutants are inducible by dexamethasone.