bluuebunny/crossref_metadata_2025 huggingface dataset:
Ubiquitin in a lower plant
Authors: POLLMANN Lutz , von KAMPEN Jan , WETTERN Michael
Published: March 2005

A detailed characterization of Chlamydomonas reinhardii cDNAs encoding ubiquitin 52‐amino‐acid fusion proteins is presented in this study. While two cDNAs (designated UBI1 and UBI3) encode the complete ubiquitin extension protein, the third one (UBI2) lacks a minor part of the 5′ region as well as a poly(A) tail. Differences between UBI1 and UBI3 are observed in the length of the poly(A) domain (13 versus 46 adenines) and in the lack of three nucleotides at the 3′ noncoding region of UBI3. According to Northern blot experiments using UBI1 as a homologous probe, at least six members of the C. reinhardii ubiquitin gene family are transcriptionally active at regular conditions. During application of severe stress (heat shock in light and darkness, and photoinhibition), the transcription of the UBI1 mRNA substantially decreases. This effect is most drastically induced by application of heat shock to illuminated cells.
Development‐dependent expression of isozymogens of monkey pepsinogens and structural differences between them
Authors: KAGEYAMA Takashi , TANABE Kazushi , KOIWAI Osamu
Published: March 2005

The developmental changes in the expression of monkey pepsinogens and structural differences between the polypeptides were investigated. Monkey pepsinogens included five different components, namely, pepsinogens A‐1–4 and progastricsin. Their respective relative levels and specific activites changed significantly during development. The sequential expression of genes for type‐A pepsinogens was particularly noteworthy. Pepsinogen A‐3 was the major zymogen at the newborn stage, accounting for nearly half of the total pepsinogens at this stage. Pepsinogen A‐2 became predominant at the 4‐month stage, and pepsinogen A‐1 predominated at the juvenile and adult stages. Enzymatic properties of pepsinogens A‐1, A‐2 and A‐3 were similar but not identical to those of pepsinogen A‐4 and progastricsin, in particular with respect to the activation processes. Each pepsin digested various protein substrates but some differences in specificity were evident. cDNA clones for five pepsinogens were isolated, and the nucleotide sequences were determined. Each cDNA contained leader, pro, and pepsin regions that encoded 15, 47, and 326 amino acid residues, respectively, with the exception of the cDNA for progastricsin in which the pro and pepsin regions encoded 43 and 329 amino acid residues, respectively. Type‐A pepsinogens exhibited a high degree of similarity, with over 96% of bases in the nucleotide sequences of the protein‐coding regions being identical. Northern analysis revealed that the level of expression of genes for type‐A pepsinogens and for progastricsin was significant at the fetal stage and increased with development.
Structural role of amino acids 99–110 in recombinant human erythropoietin
Authors: CHERN Yijuang , CHUNG Taeowan , SYTKOWSKI Arthur J.
Published: March 2005

Erythropoietin is the prime regulator of red blood cell production. Previous studies demonstrated that antipeptide antibodies to amino acids 99–119 and 111–129 bind to two non‐overlapping domains and inhibit the hormone's action (Sytkowski, A. J. & Donahue, K. A. (1987) J. Biol. Chem. 262, 1161–1165). Oligonucleotide‐directed mutagenesis now shows that amino acids 99–110 (domain 1) but not 119–129 (domain 2) are important to erythropoietin's structure and function. Mutagenesis of wild‐type human erythropoietin cDNA was used to produce a series of mutant proteins with sequential deletion of three adjacent amino acids and insertion of the sequence Glu‐Phe across the two domains. Transient expression in COS‐7 cells revealed 2.0‐kb transcripts encoded by all of the cDNAs. Domain 2 mutants exhibited specific biological activities similar to that of the wild type. In contrast, domain 1 mutants were not secreted. In vitro transcription and translation of the domain 1, domain 2 and wild‐type cDNAs resulted in the isolation of 23.5‐kDa and 32‐kDa proteins in the absence or presence of pancreatic microsomes, respectively, consistent with efficient translation of all of the mutants and equivalent post‐translational processing of each protein. The data suggest that mutation within domain 1 results in the intracellular biosynthesis of erythropoietins with altered structure, rendering them subject to rapid degradation. The bioassay of erythropoietins synthesized entirely in vitro demonstrated that domain 1 mutants were inactive, whereas both wild type and domain 2 mutant hormones exhibited biologic activity. The results are consistent with a critical role for amino acids 99–110 in the structure of human erythropoietin.
Recognition of the folding consensus in RNA secondary structures by the topological‐filtering method
Authors: BENEDETTI Giorgio , MOROSETTI Stefano
Published: March 2005

Functionally homologous RNA sequences can substantially diverge in their primary sequences but it can be reasonably assumed that they are related in their higher‐degree structures. The problem to find such structures and simultaneously satisfy as far as possible the free‐energy‐minimization criterion, is considered here in two aspects. Firstly a quantitative measure of the folding consensus among secondary structures is defined, translating each structure into a linear representation and using the correlation theorem to compare them. Secondly an algorithm for the parallel search for secondary structures according to the free‐energy‐minimization criterion, but with a filtering action on the basis of the folding consensus measure is presented. The method is tested on groups of RNA sequences different in origin and in functions, for which proposals of homologous secondary structures based on experimental data exist. A comparison of the results with a blank consisting of a search on the basis of the free energy minimization alone is always performed. In these tests the method shows its ability in obtaining, from different sequences, secondary structures characterized by a high‐folding consensus measure also when lower free energy but not homologous structures are possible. Two applications are also shown. The first demonstrates the transfer of experimental data available for one sequence, to a functionally related and therefore homologous one. The second application is the possibility of using a topological probe in the search for precise structural motifs.
Self‐catalysed affinity labeling of Qβ replicase
Authors: LINDNER Anton J. , GLASER Stephan J. , BIEBRICHER Christof K. , HARTMANN Guido R.
Published: March 2005

The spatial neighbourhood of the active center of Qβ replicase can be selectively modified by the method of self‐catalysed affinity labeling. In the template‐directed, mainly intramolecular enzymatic catalysis, the product [32P]GpG becomes specifically attached to the β subunit. Using limited digestion of the radioactively labeled polypeptide by cyanogen bromide or N‐chlorosuccinimide, we have mapped the attachment site to the region of subunit β between Trp93 and Met130. Under our reaction conditions, Lys95 is the amino acid most likely to be modified, suggesting that Lys95 lies near the nucleotide binding site in the active center.
Patchwork‐structure serpins from silkworm (Bombyx mori) larval hemolymph
Authors: SASAKI Takuji
Published: March 2005

A new serpin (serine proteinase inhibitor), having antichymotryptic activity, was isolated from silkworm, Bombyx mori, larval hemolymph and was named silkworm antichymotrypsin II (sw‐AchyII). Amino‐acid‐sequence analysis of sw‐AchyII revealed that it consisted of 375 amino acids without cysteine or glycosylated residues. sw‐AchyII formed an SDS‐undissociable complex with α‐chymotrypsin, but this complex was broken down at pH 12.5 into α‐chymotrypsin and sw‐AchyII in which the reactive site was cleaved. Amino‐acid‐sequence analysis after cleavage identified in P1‐P1′ residue at the reactive site of sw‐AchyII as Phe340‐Met341. The amino acid sequence from the amino terminus to residue 336 was completely identical to the corresponding region of sw‐AT [Takagi, H., Narumi, H., Nakamura, K. & Sasaki, T. (1990) J. Biochem. (Tokyo) 108, 372–378]. The degree of similarity between sw‐AchyII and silkworm antitrypsin (sw‐AT) from residue 337 to the carboxy terminus was only 46%. Reactive sites of both serpins were in the variable regions.
Specific inhibition of endopeptidase 24.16 by dipeptides
Authors: DAUCH Pascale , VINCENT Jean‐Pierre , CHECLER Frédéric
Published: March 2005

The inhibitory effect of various dipeptides on the neurotensin‐degrading metallopeptidase, endopeptidase 24.16, was examined. These dipeptides mimick the Pro10‐Tyr11 bond of neurotensin that is hydrolyzed by endopeptidase 24.16. Among a series of Pro‐Xaa dipeptides, the most potent inhibitory effect was elicited by Pro‐Ile (Ki∼ 90 μM) with Pro‐Ile > Pro‐Met > Pro‐Phe. All the Xaa‐Tyr dipeptides were unable to inhibit endopeptidase 24.16. The effect of Pro‐Ile on several purified peptidases was assessed by means of fluorigenic assays and HPLC analysis. A 5 mM concentration of Pro‐Ile does not inhibit endopeptidase 24.11, endopeptidase 24.15, angiotensin‐converting enzyme, proline endopeptidase, trypsin, leucine aminopeptidase, pyroglutamyl aminopeptidase I and carboxypeptidase B. The only enzyme that was affected by Pro‐Ile was carboxypeptidase A, although it was with a 50‐fold lower potency (Ki∼ 5 mM) than for endopeptidase 24.16. By means of flurimetric substrates with a series of hydrolysing activities, we demonstrate that Pro‐Ile can be used as a specific inhibitor of endopeptidase 24.16, even in a complex mixture of peptidase activities such as found in whole rat brain homogenate.
Single amino acid substitutions in the B870 α and β light‐harvesting polypeptides of Rhodobacter capsulatus
Authors: BABST Markus , ALBRECHT Hugo , WEGMANN Ines , BRUNISHOLZ René , ZUBER Herbert
Published: March 2005

To obtain information on the structural and functional role of highly conserved amino acid residues in the B870 α and β light‐harvesting polypeptides of Rhodobacter capsulatus, site‐directed mutagenesis was performed. 18 mutants with single amino acid substitutions at nine different positions in the B870 antenna polypeptides were prepared in a B800–850‐lacking strain. The characterization of the resulting phenotypes was based on a quantification of the core‐complex elements (reaction center, light‐harvesting polypeptides, bacteriochlorophyll a and carotenoid) and the core‐complex spectral characteristics (absorption maximum, absorption coefficient and fluorescence intensity). These data generally showed that strong structural effects were caused by the amino acid substitutions. Thus, the three tryptophan exchanges at the position α8 resulted in either the absence of a core complex (αTrp8→Leu), the absence of the core antenna (αTrp8→Ala) or a reduction in the carotenoid content (αTrp8→Tyr). Likewise, the mutants αPro13Gly (i.e. αPro13→Gly), βGly10Val and αPhe23Ala demonstrated an abnormal protein/pigment ratio in the core antenna, while a drastically reduced antenna size resulted from the amino acid exchange βArg45Asp. In contrast to the structural effects, the absorption maxima and the fluorescence intensities of the mutant antennae differed only slightly from the wild type. The strongest blue shift of the bacteriochlorophyll a (8 – 11 nm) was induced by substitutions of the Trp at position α43 (αTrp43→Ala, Leu or Tyr). Contrary to the other spectral effects, the absorption coefficient of bacteriochlorophyll a was strongly influenced by the amino acid substitutions and varied by 1.6‐times less (βArg45Asp) and 1.3‐times greater (αPhe25Ala) than normal. The antenna‐free mutant, αTrp8Ala, yielded a high rate of B800‐850 revertants during phototrophic growth, indicating a direct energy transfer from the B800–850 antenna to the reaction center in these strains. Although conditions for growth were generally observed to influence phenotypic expression, the structural as well as spectral effects were demonstrated to differ to the greatest extent between chemotrophically grown and phototrophically grown cells.
The secondary structure of echistatin from 1H‐NMR, circular‐dichroism and Raman spectroscopy
Authors: SAUDEK Vladimir , ATKINSON R. Andrew , LEPAGE Pierre , PELTON John T.
Published: March 2005

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine‐rich, RGD‐containing proteins, isolated from the venom of the saw‐scaled viper Echis carinatus.

Analysis of circular‐dichroism spectra indicates that, at 20°C, echistatin contains no α‐helix but contains mostly β‐turns and β‐sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40°C and 72°C being irreversible.

Raman spectra also indicate considerable β‐turn and β‐sheet (20%) structure and an absence of α‐helical structure. Three of the four disulphide bridges are shown to be in an all‐gauche conformation, while the fourth adopts a trans‐gauche‐gauche conformation.

The 1H‐NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27°C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either α‐helical or β‐sheet conformation. A number of turns could be characterised. An irregular β‐hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross‐links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.
A calorimetric study of the Ni(II)‐5′AMP system
Authors: HERRERO Luis A. , CALAFAT Antònia M. , TERRÓN Angel
Published: March 2005

The thermodynamic parameters of the interaction between Ni(II) (M) and 5′‐AMP in aqueous solution were determined calorimetrically (I= 0.1 tetramethylammonium bromide, 25°C) at physiological pH. The experimental conditions were carefully selected to avoid the polynuclear complex formation. From the Ni(5′AMP)‐complex‐formation constant (logK1= 2.55), the Ni(5′AMP)2−2‐complex‐formation constant and the Ni(5′AMP) (ML) and Ni(5′AMP)2−2 (ML2) complex‐formation enthalpies were determined (logK2= 2.34, ΔH1=–10.0 kJ/mol and ΔH2= 21.6 kJ/mol).

These results confirm the formation of the ML2 complex in solution and agree very well with the hypothesis of the ‘stacking’ between the purine rings, promoted for metal ions.
Characterization of muscle and non muscle Xenopus laevis tropomyosin mRNAs transcribed from the same gene
Authors: HARDY Serge , FISZMAN Marc Y. , OSBORNE H. Beverley , THIEBAUD Pierre
Published: March 2005

We have isolated and characterized three Xenopus laevis tropomyosin cDNAs (XTMα2, XTMα4 and XTM05). XTMα2 and XTMα4 were isolated from a stage‐42 embryo cDNA library and XTM05 from an oocyte cDNA library. XTMα2 and XTMα4 both code for a 284‐amino‐acid protein homologous to the skeletal α tropomyosin but diverge in their 3′ untranslated sequences. This divergence is due to an absence of splicing in the early embryo. XTM05 codes for a 248‐amino‐acid non‐muscle tropomyosin. Sequence analysis indicates that the three cDNAs are generated from a single gene which uses two promoters and possibly three sets of alternate exons. Northern blot analysis, with specific probes against each cDNA, was used to study the expression of the X. laevisα tropomysin gene during development and in specific tissues. This analysis showed a different temporal usage of the two promoters and characterized the tissue‐specific expression of the different transcripts.
Determination of the orientations of tryptophan analogues bound to the trp repressor and the relationship to activation
Authors: BORDEN Katherine L. B. , BECKMANN Pamela , LANE Andrew N.
Published: March 2005

The antirepressor indole 3‐propanoate has been shown by X‐ray crystallography to bind in a different orientation compared with the natural corepressor for the trp repressor, l‐tryptophan (Lawson, C. L. & Sigler, P. B. (1988) Nature 333, 869–871). This suggests a simple difference between what constitutes a corepressor versus an antirepressor.

We have used visible absorption and 1H‐NMR spectroscopy to characterise the nature of several ligand‐repressor complexes and DNA‐binding assays to assess the relative operator binding affinities. 5‐Fluorotryptophan binds with similar affinity and in the same orientation as l‐tryptophan, and is an equally effective corepressor. In contrast, the tight‐binding antirepressor indole 3‐acrylate binds in the same orientation as indole 3‐propanoate. Indole, also an antirepressor, also binds in the indole‐3‐propanoate orientation. 5‐Methyltryptamine, a corepressor, shows spectroscopic characteristics of both tryptophan and indoleacrylate, though NOEs indicate that the tryptophan orientation is preferred. These results indicate that the ammonium group in the side chain is essential both for activation and binding in the l‐tryptophan orientation. Antirepressors, lacking the ammonium group, bind in the more favourable indole‐3‐propanoate orientation.

Differences in the NMR signatures of the different repressor‐ligand complexes indicate that the details of the conformations depend on the nature of the ligands and their orientation within the binding site. Despite any conformational rearrangement of the protein on binding, dissociation of ligands is facile: 5‐fluorotryptophan dissociates rapidly at 313 K. These findings complement and extend the X‐ray and thermodynamic analyses of ligand binding.
Isolation of a novel type‐I fatty‐acid synthetase from Euglena gracilis
Authors: SIEBENLIST Ursula , WOHLGEMUTH Silvia , FINGER Karin , SCHWEIZER Eckhart
Published: March 2005

A novel, high‐molecular‐mass fatty‐acid synthetase (FAS) complex has been isolated from streptomycin‐bleached Euglena gracilis cells. The enzyme was purified 250‐fold from the crude cell homogenate and subsequently migrated upon SDS/PAGE as a single band of molecular mass 270 kDa. This apparent subunit size of the purified protein contrasted with a smaller size of only 200 kDa which was exhibited by the same protein upon immunoblotting of the crude cell extract. The purified Euglena FAS complex cosediments in a sucrose density gradient with yeast FAS and, from this, both enzymes were concluded to have the same overall molecular mass of 2.3 MDa. The enzyme described in this paper appears to be a typical type‐I FAS multienzyme which clearly differs from the E. gracilis FAS so far described. Instead, it appears to be organized structurally similar to the type‐I FAS multienzymes of lower fungi. In vitro, the purified Euglena FAS complex synthesizes mainly palmitic acid, or its CoA ester, from acetyl CoA and substrates. The Km values for acetyl CoA and malonyl CoA are 20 μM and 31 μM, respectively. Similar to the FAS enzymes of other lower eucaryotes, the Euglena type‐I FAS is a flavoprotein. In contrast to yeast FAS, however, the flavin cofactor appears to be covalently attached to the enzyme protein. By immunological techniques, the enzyme was shown to be absent in green as well as in etiolated E. gracilis cells, while being rapidly induced upon streptomycin bleaching of heterotrophically growing green cells. The data suggest an inverse correlation between organellar development and derepression of this FAS complex.
Purification and characterization of two xylanases from Trichoderma longibrachiatum
Authors: ROYER John C. , NAKAS James P.
Published: March 2005

Two endoxylanases were purified from the culture medium of Trichoderma longibrachiatum. Both enzymes were highly basic, and lacked activity on carboxymethyl‐cellulose. An enzyme of 21.5 kDa (xylanase A) had a specific activity of 510 U/mg protein, a Km of 0.15 mg soluble xylan/ml, possessed transglycosidase activity and generated xylobiose and xylotriose as the major endproducts from xylan or xylose oligomers. A larger enzyme of 33 kDa (xylanase B) had a specific activity of 131 U/mg protein, a Km of 0.19 mg soluble xylan/ml, lacked detectable transglycosidase activity and generated xylobiose and xylose as major endproducts from xylan and xylose oligomers. Xylotriose was the smallest oligomer attacked by both enzymes. In addition, xylotriose inhibited hydrolysis of xylopentanose by both enzymes, while xylobiose appeared to inhibit xylanase B, but not xylanase A.
Heparinase II from Flavobacterium heparinum
Authors: MOFFAT Colin F. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B.
Published: March 2005

Saccharides produced by the action of heparinase II on native pig mucosal heparin (heparin Is), de‐N‐sulphated heparin (heparin Ih), N‐acetylheparin (heparin Ia), de‐N/O‐sulphated heparin (heparin IVh), de‐O‐sulphated heparin (heparin IVs) and de‐O‐sulphated N‐acetylheparin (heparin IVa) were analysed by reversed‐phase HPLC using Spherisorb ODS2. Fractions obtained by gel filtration with Bio‐Gel P‐4 were similarly examined.

Heparin Is gave ΔUA‐2S → GlcNS‐6S (Is) as the major unsaturated disaccharide and lesser amounts of ΔUA → GlcNS‐6S (IIs), ΔUA‐2S → GlcNS (IIIs), ΔUA → GlcNS (IVs), ΔUA‐2S → GlcNAc‐6S (Ia), ΔUA → GlcNAc‐6S (IIa), ΔUA‐2S → GlcNAc (IIIa) and ΔUA → GlcNAc (IVa).

Heparins Ia, IVa and IVs gave as the predominant unsaturated disaccharide that corresponding to the major repeat structure of the polymer. These were respectively ΔUA‐2S → GlcNAc‐6S (Ia), ΔUA → GlcNAc (IVa) and ΔUA → GlcNS (IVs). Minor disaccharides from the heterogeneous structure in native pig heparin and from residual O‐sulphates after the de‐O‐sulphating process were detected.

Heparin Ih was degraded more slowly than any of the N‐substituted heparins. The predominant unsaturated disaccharide was Ih, which was derived from the major repeating unit. In addition, disaccharides IIh, IIIh, Ia, IIa and IVa were detected.

Heparin IVh showed little degradation, the unsaturated disaccharide IVh not being detected after 24 h. Disaccharide IVa was obtained from the heterogeneous sequence in heparin IVh.

Several higher oligosaccharides were identified in the gel‐filtration fractions including saccharides from the linkage region (for heparin Is and IVa) and the anti‐thrombin binding site (for heparin Is only). A tetrasaccharide and hexasaccharide, with the structures ΔUA → GlcNAc → UA → GlcNAc and ΔUA → GlcNAc → UA → GlcNAc → UA → GlcNAc, were present in the HPLC profiles of heparins Ia and IVa.
Volume changes in high‐affinity calcium binding of the sarcoplasmic reticulum calcium‐transport enzyme
Authors: STEPHAN Sibylle , HASSELBACH Wilhelm
Published: March 2005

The effect which hydrostatic pressure exerts on the hydrolysis of dinitrophenyl phosphate and nitrophenyl phosphate by the sarcoplasmic reticulum calcium‐transport enzyme was determined. Activation volumes for substrate hydrolysis at saturating and non‐saturating concentrations of calcium were determined and used to evaluate volume increments for initial calcium‐binding. A reaction scheme in which two unidirectional substrate‐driven reactions transfer high‐affinity into low‐affinity calcium‐binding sites was applied to determine binding‐volume increments. It has been inferred from the pressure dependence of the volume‐generating function, defined as the difference between the reciprocal reaction rates of the saturated and the unsaturated enzyme, that calcium binding proceeds in two steps. The two associated binding constants are endowed with large binding‐volume increments of opposite signs (+ 84 to + 207 ml/mol and − 3 to − 136 ml/mol). Under different experimental conditions, with respect to the temperature, degree of calcium saturation and absence or presence of Me2SO, they add up to the same integral volume increment of 73 ± 3.5 ml/mol for the entry of two calcium ions into the reaction cycle. In aqueous media, the two binding constants contribute about equally to binding and to the observed binding‐volume increment. The presence of Me2SO strongly favours the first binding step. The size of the integral volume increment is in line with that determined for the interaction of calcium with calmodulin [Kupke, D. W. & Dorrier, T. E. (1986) Biochem. Biophys. Res. Commun. 38, 199–204].
Disulfide‐bonded dimerization of fibronectin in vitro
Authors: VARTIO Tapio , KUUSELA Pentti
Published: March 2005

Human plasma fibronectin was denatured with 8 M urea and reduced with dithiothreitol. Dialysis or dilution of the solution led to formation of fibronectin dimers which migrated in non‐reducing SDS/PAGE similarly to untreated control protein. When the redimerized fibronectin was reduced and re‐electrophoresed it formed a doublet of α and β chains of equal intensity indicating that it was a heterodimer. Low concentrations (< 1 nM) of Fe3+ enhanced the redimerization of fibronectin, suggesting that metal ions may mediate oxidative reactions in the formation of the disulfides. Consequently, redimerization of fibronectin was completely prevented by deferoxamine, an iron chelator. Dimerization of fibronectin took place most effectively at pH ≥ 8.8 but decreased strongly at lower pH, representing more unfavourable conditions for the action of the thiolate anion in the thiol/disulfide exchage reaction. Redimerized fibronectin, however, lost many of its binding properties to macromolecular ligands, suggesting that the disulfide bonding did not entirely regenerate the proper conformation of the protein. Pulse/chase experiments of fibroblast cultures showed that the initially monomeric fibronectin was rapidly and quantitatively dimerized under conditions representing natural pH and environment. SDS/PAGE analysis of the dialyzed urea‐denatured/reduced thrombin and plasmin digests of fibronectin revealed that the NH2‐terminal 30‐kDa fragment and other fragements that contained intrachain disulfides quantitatively regained their non‐reduced electrophoretic mobility. The results show that the dimerization and formation of intrachain disulfides of fibronectin may occur, in part, spontaneously, based on the amino acid sequence information of the protein. However, complete disulfide formation may also need other factors, present only in living cells, as suggested by pulse/chase experiments in fibroblasts.
Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria
Authors: RASMUSSON Allan G. , MØLLER Ian M.
Published: March 2005

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone‐insensitive internal oxidation of NADPH by inside‐out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD+‐dependent and NADP+‐dependent malate dehydrogenases were affected by the addition of Ca2+.

The pH dependence and kinetics of Ca2+‐dependent NADPH oxidation by inside‐out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2.

Half‐maximal stimulation of the NADPH oxidation was found at 3 μM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane‐bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+.

Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone‐insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.
Functional modification by cholera‐toxin‐catalyzed ADP‐ribosylation of a guanine‐nucleotide‐binding regulatory protein serving as the substrate of pertussis toxin
Authors: IIRI Taroh , OHOKA Yoshiharu , UI Michio , KATADA Toshiaki
Published: March 2005

The α subunits of Gi (Giα) and Gs (guanine‐nucleotide‐binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP‐ribosylated by cholera toxin in differentiated HL‐60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N‐formylmethionine). The ADP‐ribosylation site of Giα modified by cholera toxin appeared to be different from that modified by pertussis toxin [Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M. & Katada, T. (1989) J. Biol. Chem. 264, 21394–21400]. This allowed us to investigate how the two types of ADP‐ribosylation influence the function of the signal‐coupling protein. The major findings observed in HL‐60 cell membranes, where the same Giα molecule was ADP‐ribosylated by treatment of the membranes with either toxin, are summaried as follows. (a) More fMLF bound with a high affinity to cholera‐toxin‐treated membranes than to the control membranes. The high‐affinity binding was, however, not observed in pertussis‐toxin‐treated membranes. (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis‐toxin‐treated membranes. In contrast, fMLF‐dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera‐toxin‐treated membranes. (c) Giα, once modified by cholera toxin, still served as a substrate of pertussis‐toxin‐catalyzed ADP‐ribosylation; however, the ADP‐ribosylation rate of modified Gi was much lower than that of intact Gi. These results suggested that Gi ADP‐ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high‐affinity fMLF receptors, and that hydrolysis of GTP bound to the α subunit was selectively impaired by its ADP‐ribosylation by cholera toxin. Thus, unlike the ADP‐ribosylation of Gi by pertussis toxin, cholera‐toxin‐induced modification would be of great advantage to the interaction of Gi with receptors and effectors that are regulated by the signal‐coupling protein. This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal‐transduction systems.
Primary structure and processing of the Candida tsukubaensisα‐glucosidase
Authors: KINSELLA B. Therese , HOGAN Stephane , LARKIN Angela , CANTWELL Barbara A.
Published: March 2005

The nucleotide sequence of a 4.39‐kb DNA fragment encoding the α‐glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the α‐glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N‐terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the α‐glucosidase precursor is proteolytically processed by removal of an N‐terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63–65 kDa and 50–52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expresion in the heterologus host, the C. tsukubaensisα‐glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensisα‐glucosidase, the rabbit sucrase‐isomaltase complex (proSI) and human lysosomal acid α‐glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.
Sequence‐specific complex formation of DNA and a eukaryotic sequence‐specific endonuclease, SceI
Authors: KAWASAKI Katsumi , TAKAHASHI Mitsuhiro , ANDO Tadahiko , SHIBATA Takehiko
Published: March 2005

Endo ·SceI is a eukaryotic sequence‐specific endonuclease of 120 kDa that causes sequence‐specific double‐stranded scission of DNA. Unlike results with restriction enzymes, we found a consensus sequence around the cleavage sites for Endo ·SceI instead of a common sequence. We searched for conditions for studying the binding of Endo ·SceI to DNA other than cutting. Under optimized conditions including gel mobility shift assay, Endo ·SceI exhibited sequence‐specific binding to a short double‐stranded DNA (41 base pairs) containing a cleavage site and the DNA reisolated from the protein‐DNA complex was not cleaved. The analysis of the complex of Endo ·SceI and DNA isolated by the gel mobility shift experiments showed that the DNA‐binding entity in the Endo ·SceI preparation does have Endo ·SceI activity and consists of an equal amount of 75‐kDa and 50‐kDa polypeptides. Based on this observation and those from previous studies, we conclude that Endo ·SceI is a heterodimer of the 75‐kDa and 50‐kDa subunits. Under the present assay conditions, Endo ·SceI did not show binding to single‐stranded DNA having the same sequence of either plus or minus strand of the double‐stranded DNA containing the cleavage site (the 41‐bp DNA). Endo ·SceI showed significantly higher affinity for the consensus sequence than the major cleavage site in pBR322 DNA. Unlike the cleavage of DNA by Endo ·SceI which requires Mg2+, this sequence‐specific binding is independent of but stimulated by Mg2+.
Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensisδ‐endotoxins
Authors: ODDOU Pascale , HARTMANN Herbert , GEISER Martin
Published: March 2005

To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand‐blot experiments with the three activated CryIA toxins. The CryIA(a) and the CryIA(b) toxins bind the same 170‐kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170‐kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA(a) or CryIA(b) toxin. This indicates that the sugars are not important for the binding of these toxins. A model for a protein complex binding the B. thuringiensis HD1 ICPs is presented. Our results support the idea that binding proteins on membranes of the gut epithelial cells of H. virescens larvea are important for the specificity of the bacterial toxins.
Characterization and distribution of cis‐prenyl transferase participating in liver microsomal polyisoprenoid biosynthesis
Authors: ERICSSON Johan , THELIN Anders , CHOJNACKI Tadeusz , DALLNER Gustav
Published: March 2005

The properties of rat liver cis‐prenyl transferase, mediating the synthesis of polyisoprenoid pyrophosphate from trans, trans‐farnesyl pyrophosphate and [3H]isopentenyl pyrophosphate were studied. The Km values for farnesyl pyrophosphate and isopentenyl pyrophosphate were found to be 25 μM and 4.4μM, respectively. Appropriate conditions were established to measure the condensation reaction, which was linear during the first hour using 1 mg microsomal protein. Various detergents could solubilize the enzyme, but the presence of Triton X‐100 was required during the incubation to obtain full activity. There was also an absolute requirement for Mg2+ and the pH maximum was 7.0. Inorganic phosphate, especially pyrophosphate, proved to be inhibitory. cis‐Prenyl transferase is associated mainly with the cytoplasmic surface of rough microsomes and, to some extent, also with smooth I microsomes, but was almost absent from smooth II microsomes. At all localizations, the product is polyprenyl pyrophosphate and to some extent, also polyprenyl monophosphate. The isoprenoids formed contain 15–18 units in the presence of detergents and 16–20 units in the absence of detergents.
Characterization of a camptothecin‐resistant human DNA topoisomerase I in an in vitro system for Simian virus 40 DNA replication
Authors: ISHIMI Yukio , NISHIZAWA Miwako , ANDOH Toshiwo
Published: March 2005

DNA topoisomerase I was required for bidirectional DNA replication in an in vitro system for Simian virus 40 (SV40) DNA replication with purified proteins in which the replication fork moved at the rate of 260 nucleotides/min on average. DNA topoisomerase I purified from camptothecin‐resistant human lymphoblastoid cells, which confers high resistance of cellular DNA replication to camptothecin [Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y. & Okada, K. (1987) Proc. Natl Acad. Sci. USA 84, 5565–5569], was characterized using this system. The activity of stimulating bidirectional DNA replication was comparable between two topoisomerase I from parental and resistant cells, i.e. in its dose‐response relationship and in its time course for DNA synthesis.

Camptothecin severely inhibited the leading as well as the lagging strand synthesis in the reaction containing the wild type topoisomerase I but not the mutant type topoisomerase I. The mutant type topoisomerase I was over 125‐fold as resistant to camptothecin as the wild type topoisomerase I. These results are in good agreement with those on the sensitivity of cellular DNA synthesis to camptothecin in the resistant cells. These findings suggest that topoisomerase I is involved in cellular DNA replication as a swivelase and the mutation conffering camptothecin‐resistance on the enzyme does not affect its functional efficiency in this system.
5′‐Nucleotidase from the electric ray electric lobe
Authors: VOLKNANDT Walter , VOGEL Manfred , PEVSNER Jonathan , MISUME Yoshio , IKEHARA Yukio , ZIMMERMANN Herbert
Published: March 2005

A cDNA encoding a 5′‐nucleotidase was identified by screening a λgt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63833 Da. The N‐terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C‐terminus contains a stretch of hydrophobic amino acids, considered to be cleaved on post‐translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N‐linked glycosylation sites. Electric ray 5′‐nucleotidase shares 61% amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5′‐nucleotidase activity and also additional enzyme activities like UDP‐glucose hydrolase. Polyclonal antibodies raised against 5′‐nucleotidase from mammalian sources or the electric ray electric organ reveal mutual crossreactivity. Interestingly, there are 5–7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5′‐nucleotidase, 3′‐nucleotidase or phosphodiesterase activity. 5′‐nucleotidase isolated from Torpedo electric organ hydrolyzes UDP‐glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5′‐nucleotidase from multifunctional nucleotide hydrolases is discussed.
Lipoamide Dehydrogenase from Azotobacter vinelandii: site‐directed mutagenesis of the His450‐Glu455 diad
Authors: BENEN Jacques , van BERKEL Willem , ZAK Zdzislaw , VISSER Ton , VEEGER Cees , de KOK Arie
Published: March 2005

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced by other residues. His450, the active‐site base, was changed into Ser, Tyr and Phe. Pro451, in cis conformation, was changed into Ala. Glu455 was replaced with Asp and Gln.

Absorption, fluorescence and CD spectroscopy of the mutated enzymes in their oxidized state (Eox) showed only minor changes with respect to the wild‐type enzyme, wherease considerable changes were observed in the spectra of the two‐electron‐reduced (EH2) species of the enzymes upon reduction by the substrate dihydrolipoamide. Differences in extent of reduction of the flavin by NADH indicate that the redox potential of the flavin is altered by the mutations. Enzyme Pro455 Ala showed the greatest deviation from wild type. The enzyme is very easily over‐reduced to the four‐electron reduced state (EH4) by dihydrolipoamide. This is probably due to a change in the backbone conformation caused by the cis‐trans conversion.

From studies on the pH dependence of the thiolate charge‐transfer absorption and the relative fluorescence of EH2 of the enzymes, it is concluded that mutation of His450 results in a relatively simple and easily interpreted distribution of electronic species at the EH2 level. For all three His450‐mutated enzymes an apparent pKa1 near 5.5 is calculated that is assigned to the interchange thiol. A second apparent pKa2 is calculated of 6.9, 7.5 and 7.1 for the His450 Phe, ‐Ser and ‐Tyr enzymes, respectively, and signifies the deprotonation of the tautomeric equilibrium between the interchange and charge‐transfer thiols. The difference in apparent pK2 values between the His45‐mutated enzymes is explained by changes in micropolarity.

At the EH2 level the wild‐type enzyme consists of multiple electronic forms as reported for the Escherichia coli enzyme [Wilkinson, K. D. and Williams C. H. Jr (1979) J. Biol. Chem. 254, 852–862]. Based on the results obtained with the His450‐mutated enzymes, it is concluded that the lowest pKa is associated with the interchange thiol. A model for the equilibrium species of the wild‐type enzyme at the EH2 level is presented which takes three pKa values into account.

The results of the pH dependence of the electronic species at the EH2 level of Glu455‐mutated enzymes essentially follow the model proposed for the wild‐type enzyme. However mutation of Glu455 shifts the tautomeric equilibrium of EH2 in favor of the charge‐transfer species. This effect is explained by impaired ion‐pair formation between the protonated His and the interchange thiolate and a lowering of the pka of His450.
An 100‐kDa arachidonate‐mobilizing phospholipase A2 in mouse spleen and the macrophage cell line J774
Authors: WIJKANDER Jonny , SUNDLER Roger
Published: March 2005

A phospholipase A2 hydrolyzing arachidonic‐acid‐containing phospholipids has been purified 5600‐fold from mouse spleen and to near homogeneity from the macrophage cell line J774. A molecular mass of 100 kDa for the enzyme was estimated by SDS/PAGE, while it migrated as a 70‐kDa protein upon gel chromatography. The enzyme from both sources showed the same characteristics as that previously identified in murine peritoneal macrophages [Wijkander, J. & Sundler, R. (1989), FEBS Lett. 244, 51–56], i.e. it was totally dependent on Ca2+ with half‐maximal activity at approximately 0.7 μM and hydrolyzed arachidonoyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol equally well. Also, the platelet‐activating‐factor precursor, 1‐O‐alkyl‐2‐arachidonoylglycerophosphocholine, was hydrolyzed to a similar extent. A preference for arachidonoylphosphatidylcholine over oleoylphosphatidylcholine was seen both with sonicated vesicles and labeled macrophage membranes as substrate.

Ca2+‐dependent interaction of the enzyme with sonicated vesicles composed of neutral phospholipids led to rapid initial hydrolysis, followed by loss of catalytic activity. Such inactivation did not occur with vesicles of pure anionic phospholipids, or with membranes prepared from macrophages.

Phospholipase A2, purified from J774 cells, was rapidly phosphorylated by protein kinase C type‐II, leading to incorporation of approximately 0.5 mol phosphate/mol enzyme.
Gene sequence of mouse B‐type proline‐rich protein MP4
Authors: ROBERTS Stefan G. E. , LAYFIELD Roy , BANNISTER Andrew J. , McDONALD Charles J.
Published: March 2005

A mouse genomic B‐type proline‐rich protein (PRP) cosmid clone was isolated by cDNA hybridisation and mapped, the gene region was subcloned and 3770 bp were sequenced. This gene (MP4) contained three introns and encoded a 1020‐nt (nt, nucleotide) mRNA for a PRP precursor 300 amino acids long arranged with 11 imperfect 18‐residue proline‐rich repeats. The transcriptional start point was determined by S1 nuclease mapping and primer extension to be 26 bp downstream of a TATAA sequence. Sequence comparisons revealed that only two regions from positions ‐650 bp ––30 bp were highly conserved in all other PRP genes, PRP boxes 1 and 2. Box 1 at positions ‐112 to ‐135 contained ets‐like and rel/NFkB‐like elements and was 74% conserved over 23 bp. Box 2 at positions ‐33 –– 51 was 53% conserved over 19 bp. A search of the EMBL and GenBank sequence libraries indicated that PRP box 1 was only present upstream of the known mammalian PRP gene sequences and was absent from other genes. These conserved sequences may thus be relevant to the tissue‐specific and β‐adrenergic regulation of PRP gene transcription.
Cloning, sequencing and expression studies of the genes encoding amicyanin and the β‐subunit of methylamine dehydrogenase from Thiobacillus versutus
Authors: UBBINK Marcellus , van KLEEF Mario A. G. , KLEINJAN Dirk‐Jan , HOITINK Carla W. G. , HUITEMA Fienke , BEINTEMA Jaap J. , DUINE Johannis A. , CANTERS Gerard W.
Published: March 2005

The genes encoding amicyanin and the β‐subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyanin is involved in electron transport from methylamine to oxygen. The amino acid sequence deduced from the nucleotide sequence of the amicyaninencoding gene is in agreement with the published protein sequence. The gene codes for a pre‐protein with a 25‐amino‐acid‐long signal peptide. The amicyanin gene could be expressed efficiently in Escherichia coli. The protein was extracted with the periplasmic fraction, indicating that pre‐amicyanin is translocated across the inner membrane of E. coli. Sequence studies on the purified β‐subunit of MADH confirm the amino acid sequence deduced from the nucleotide sequence of the corresponding gene. The latter codes for a pre‐protein with an unusually long (56 amino acids) leader peptide. The sequencing results strongly suggest that pyrroloquinoline quinone (PQQ) or pro‐PQQ is not the co‐factor of MADH.