bluuebunny/crossref_metadata_2025 huggingface dataset:
Carboxylic acid reductase: a new tungsten enzyme catalyses the reduction of non‐activated carboxylic acids to aldehydes
Authors: WHITE Hiltrud , STROBL Gerd , FEICHT Richard , SIMON Helmut
Published: March 2005

An enzyme which we call carboxylic acid reductase (aldehyde dehydrogenase) seems to be the first which is able to reduce non‐activated carboxylic acids to aldehydes at the expense of reduced viologens. There is no further reduction of the aldehydes to the corresponding alcohols. In the presence of oxidized viologens aldehydes can be dehydrogenated to carboxylic acids roughly 20 times faster than the latter are reduced. The specific enzyme activity in crude extracts is about 100 times increased if 10 μM tungstate and a sulphur source in addition to sulphate is given to the growth medium of Clostridium thermoaceticum.

Carboxylic acid reductase seems to be present in two forms. One has an apparent molecular mass of about 240 kDa and is bound to red‐Sepharose, whereas, the other, a form of an apparent molecular mass of about 60 kDa, is not bound. SDS gel electrophoresis shows a higher complexity.

The very labile enzyme has been enriched by a factor of about 145 by binding to octyl‐Sepharose and further chromatographic separation by red‐Sepharose and FPLC using Mono‐Q and phenyl‐Superose columns. After cell growth in the presence of [185W]tungstate, radioactivity coincides with the two forms of enzyme activity during all purification steps. This is also the case when the enzyme is electrophoretically separated on polyacrylamide slab gels.
Assay and purification of the adenosylcobalamin‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri
Authors: MICHEL Christoph , HARTRAMPF Günther , BUCKEL Wolfgañg
Published: March 2005

A continuous spectrophotometric assay was developed for the adenosylcobalamin–dependent 2‐methyleneglutarate mutase from Clostridium barkeri. Thereby the product (R)‐3‐methylitaconate is converted by the Δ‐isomerase from the same organism to 2,3‐dimethylmaleate which absorbs at 240 nm, much higher than both parent compounds (Δɛ= 3.7 mM−1· cm−1). In addition a discontinuous assay using the facile formation of 2,3‐dimethylmaleic anhydride in aqueous solution at pH 0–1 (Δɛ= 4.0 mM−1· cm−1 at 256 nm) was established. The mutase and the isomerase were purified together by chromatography on quaternary‐amine–Sepharose (Q‐Sepharose) and on cyanocobalamin‐agarose. The enzymes were separated and obtained in homogenous forms by preparative PAGE in non‐denaturing buffer. Both enzymes appear to be homotetramers with subunits of 70 kDa (mutase) and 50 kDa (isomerase). The equilibrium constants for both reactions were determined at I= 0.1 M and 25°C: K1,app= [(R)‐3‐methylitaconate] · [2‐methyleneglutarate]−1= 0.26 ± 0.04, K2,app= [2,3‐dimethylmaleate] · [(R)‐3‐methylitaconate]−1= 7.40 ± 0.21
Post‐translational modifications in mitotic yeast cells
Authors: NEVALAINEN Leena T. , LOUHELAINEN Jari , MAKAROW Marja
Published: March 2005

We have recently shown that secretion of invertase is not inhibited in the yeast Saccharomyces cerevisiae during mitosis, but continues, as during interphase. This is in contrast with the mammalian cell, where membrane traffic stops at the onset of prometaphase. Here we extend our findings by showing that the bulk of the cell surface glycoproteins and mannans, as well as the yeast pheromone α‐factor, traverse the sccretory pathway during mitosis. We show that the mitotic cells are able to carry out several types of post‐translational modification of secretory proteins. (a) The secretory protein invertase was oligomerized and extensively glycosylated, (b) the N‐glycan cores of bulk‐cell surface mannans were extended with outer chains, (c) some N‐glycans were phosphorylated, (d) the protein‐bound O‐glycans were extended up to tetramannosides, (e) prepro‐ka‐factor was proteolytically processed to α‐factor molecules. We conclude that the secretory pathway in yeast remains fully functional throughout the cell cycle.
Localization of the ATP‐binding site in the 23‐kDa and 20‐kDa regions of the heavy chain of the skeletal muscle myosin head
Authors: MARUTA Shinsaku , MIYANISHI Takayuki , MATSUDA Genji
Published: March 2005

Three kinds of ATP analogues were synthesized. These ATP analogues can be classified into two conformations, i.e. syn and anti forms with respect to the N‐glycosidic bond between adenine and ribose groups of ATP. 3′‐O‐(N‐Methylanthraniloyl)‐2‐azidoadenosine 5′‐triphosphate (MantN23ATP) is recognized as the anti form, as ATP, and the other two, 3′‐O‐(N‐methylanthraniloyl)‐8‐azidoadenosine 5′‐triphosphate (MantN83ATP) and 1, N6′‐etheno‐8‐azidoadensine 5′‐triphosphate (ɛN83ATP) are both syn forms. Mant and etheno groups are both fluorescent which allows detection of their binding to proteins. The photochemical binding of azido groups in ATP análogues to the myosin active site, examined in the presence and absence of ATP, showed that all the analogues to the site of myosin ATPase. These analogues also acted as substrates of the ATPase and were hydrolyzed in the active site, as judged by competitive inhibition of the ATPase and by their ATPase activities. Of these analogues, MantN23ATP is very similar to ATP in divalent‐cation dependence of its hydrolysis rate and in its ability to trap ADP in the active site with vanadate, while the other two are different from ATP in these respects. The photochemical binding sites of ATP analogues were localized by gel electrophoresis of trypsinized myosin ATPase with photocross‐linked ATP analogues and/or by isolating the modified peptides. MantN23ATP was found in the 23‐kDa fragment which has a structure common to ATP‐binding proteins, i.e. Gly‐Xaa‐Gly‐Xaa‐Gly‐Lys‐Thr. Mant N83ATP was found in a region of the 20‐kDa fragment where actin is reported to attach.
Post‐transcriptional regulation of 5S rRNA synthesis following myoblast differentiation
Authors: ZAHRADKA Peter , LARSON Dawn E. , SELLS Bruce H.
Published: March 2005

Terminal differentiation of proliferating rat L6 myoblasts to syncytial multi‐nucleated myotubes results in a decline in the cellular levels of 5S rRNA. This decrease parallels the drop in ribosome number and the reduction in 45S rRNA precursor gene transcription previously documented [Jacobs et al. (1985) Eur. J. Biochem. 150, 255]. The activity of RNA polymerase III in 100000 ×g supernatants from myoblasts and myotubes in similar suggesting the level of this enzyme does not limit 5S rRNA synthesis. Nuclear and cell‐free transcription assays were used to measure 5S rRNA synthesis in myoblasts and myotubes. No differences were observed in the rate of 5S rRNA gene transcription, thus demonstrating that 5S rRNA levels in myotubes are not transcriptionally controlled. To determine whether RNA stability plays a role, the half‐life of newly synthesized 5S rRNA was measured in vivo. The half‐life of nuclear 5S rRNA was calculated to be 370 min and 65 min in myoblasts and myotubes, respectively, demonstrating that the decrease in 5S rRNA levels following terminal differentiation is regulated post‐transcriptionally.
Crystallization properties and structure of Panulirus interruptus haemocyanin
Authors: WICHERTJES Trijntje , KEEGSTRA Wilko , NEUTEBOOM Ben , HAZES Bart , BEINTEMA Jaap J. , van BRUGGEN Ernst F. J.
Published: March 2005

Electron‐microscopic studies revealed that two types of subunits of Panulirus interruptus haemocyanin crystallize in different ways. Homohexamers of subunit a give close‐packed two‐dimensional crystals whereas homohexamers of subunit c form open two‐dimensional arrays. We applied computer‐image analysis to these arrays and studied the differences in crystallization properties by combining the electron‐microscopic data with amino acid sequence information and the X‐ray diffraction model of subunit a.
Purification and characterization of a nuclear factor which binds specifically to the upstream activation sequence of Saccharomyces cerevisiae enolase 1 gene
Authors: MACHIDA Masayuki , JIGAMI Yoshifumi , TANAKA Hideaki
Published: March 2005

The nuclear factor which specifically binds to the upstream activation sequence (UAS) of the enolase 1 gene (ENO1) of yeast Saccharomyces cerevisiae was purified by sequence‐specific affinity chromatography. The purified factor gave two closely migrated bands at 32 kDa on SDS/PAGE. The binding activities were eluted from a gel filtration column at molecular masses of 110 kDa and 60 kDa, suggesting a dimeric and a tetrameric assembly of the factor in the native form. The region protected by the purified factor against deoxyribonuclease I digestion contained the sequence ACCCAAACACC which is highly similar to the consensus sequence present in the 5′‐flanking region of the ribosomal protein genes (RPG box). We also identified the other factor specific to the ENO1 UAS which gave a single peak at a molecular mass of 120 kDa in gel filtration. We suggest the existence of multiple binding to the ENO1 UAS by at least two factors: one is the factor which we purified with a molecular mass of 32 kDa on SDS/PAGE and the other is the factor like RAP1 protein which generally recognizes the RPG‐box‐like sequence.
Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi.V) human erythrocytes
Authors: VIGNAL Alain , RAHUEL Cécile , EL MALIKI Bouchra , LONDON Jacqueline , LE VAN KIM Caroline , BLANCHARD Dominique , ANDRE Catherine , d'AURIOL Luc , GALIBERT Francis , BLAJCHMAN Morris A. , CARTRON Jean‐Pierre
Published: March 2005

In the Miltenberger class V (Mi.V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossing‐over between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti‐GPA antibodies specific for discrete domains of GPA, it was found that the Mi.V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N‐terminus.

As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in λEMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A‐1–A‐7) and five exons (B‐1–B‐5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi.V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi.V glycoprotein gene was composed of exon B‐1 of the GPB gene followed by exons A‐2 and A‐3 of the GPA gene and the exons B‐3, B‐4 and B‐5 of the GPB gene. Exon B‐1 can be distinguished from exon A‐1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N‐terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A‐3 and exon B‐3 was confirmed by amplification of the DNA region where the putative crossing‐over has occurred and it was deduced that the Mi.V glycoprotein is a hybrid molecule composed of amino acid residues 1–58 from GPA fused to amino acid residues 27–72 of GPB. In addition, the finding that part of the signal peptide and the 5′‐untranslated region are derived from GPB suggests that the genetic background of the Mi.V variant is rather complex and may involve a cascade of recombination or gene conversion events.
NMR study of a 34‐residue N‐terminal fragment of the parathyroid‐hormone‐related protein secreted during humoral hypercalcemia of malignancy
Authors: BARDEN Julian A. , KEMP Bruce E.
Published: March 2005

The proton resonances of the biologically active peptide parathyroid‐hormone‐related protein (residues 1–34) were assigned using one‐dimensional spin‐decoupling techniques, two‐dimensional correlated spectroscopy and by comparing the spectra of the peptides 1–20, 1–25, 1–29, 7–34 and 15–34. The conformation of 1–34 was determined using one‐ and two‐dimensional nuclear Overhauser enhancement spectroscopy in the rotating frame. Amide proton temperature coefficients, vicinal coupling constants and circular dichroic spectra helped reveal a surprisingly compact structure with residues 3–9 forming α‐helix, type‐I β‐turns between residues 10–13 and 16–19 and several interactions between the N‐terminal residues and the C‐terminal residues. Of these latter, the strongest appeared to be between Asp‐10 and Phe‐22. One peptide surface in the deduced model presents multiple positive charges, while the opposite surface has a hydrophobic character possibly functioning to exclude water from the binding interface and enhancing the binding constant.
Stimulation of the methylcoenzyme M reduction by uridine‐5′‐diphospho‐sugars in cell‐free extracts of Methanobacterium thermoautotrophicum (strain ΔH)
Authors: KELTJENS Jan T. , KRAFT Harry J. , DAMEN Wim G. , van der DRIFT Chris , VOGELS Godfried D.
Published: March 2005

The hydrogen‐dependent reduction of methylcoenzyme M catalyzed by coenzyme‐depleted cell‐free extracts of Methanobacterium thermoautotrophicum was stimulated by micromolar concentrations of a UDP‐disaccharide present in the organism. The compound was isolated and identified as UDP‐1‐O‐α‐D‐2‐acetamido‐2‐deoxygluco‐pyranose (UDPGlcpNAc) glycosidically linked to 2‐acetamido‐2‐deoxymannopyranosyluronic acid. Maximal stimulation was observed when both the UDP‐disaccharide and mercaptoheptanoylthreonine phosphate were present in the reaction mixtures. The UDP derivative isolated was not specific in its action: other UDP‐sugars tested in micromolar concentrations stimulated the methylcoenzyme M reduction to the same extent. The activated sugars presumably substitute for ATP, which is usually required in much higher concentrations to activate the methylcoenzyme M reductase system.
Presence of fucosylated triantennary, tetraantennary and pentaantennary glycans in transferrin synthesized by the human hepatocarcinoma cell line Hep G2
Authors: CAMPION Bruno , LÉGER Didier , WIERUSZESKI Jean‐Michel , MONTREUIL Jean , SPIK Geneviève
Published: March 2005

Transferrin synthesized by a human hepatocellular carcinoma cell line Hep G2 (called Hep G2 transferrin) was purified from culture media by immunoaffinity chromatography on a rabbit anti‐(human serotransferrin) IgG column. The eluted transferrin was then resolved into five peaks on a cation‐exchange column using the fast protein liquid chromatography system. The major fraction, named Hep G2 transferrin fraction C, having a molecular mass of 82.5 kDa was found to be homogeneous in polyacrylamide gel electrophoresis and in concanavalin‐A‐affinity crossed immunoelectrophoresis.

A comparative analysis of the molar carbohydrate composition of normal human serotransferrin and of Hep G2 transferrin fraction C shows an increase in the latter in the number of galactose and N‐acetylglucosamine residues and in the presence of fucose, which is absent in normal transferrin.

By a combination of methylation analysis and NMR spectroscopy, the primary structure of the oligosaccharide alditols released from Hep G2 transferrin fraction C by reductive alkaline cleavage has been established as triantennary, tetraantennary and pentaantennary N‐acetyllactosaminic structures with fucose residues (α1–3)‐linked to peripheral N‐acetylglucosamine residues.

These results indicate that the increase in the number of antennae in transferrin glycans synthesized by the hepatocarcinoma cell line is much more pronounced than in liver diseases such as alcoholic cirrhosis and that in addition, the malignant transformation of human liver induces the presence of fucose.
Conformational consequences of the incorporation of arabinofuranosylcytidine in DNA
Authors: PIETERS Jane M. L. , de VROOM Erik , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

The self‐complementary octamers d(CGCTAGCG) and d(CGaCTAGCG) (aC, arabinofuranosylcytidine) were studied by means of NMR spectroscopy. It is shown that d(CGaCTAGCG), under suitable conditions of oligonucleotide concentration, ionic strength and temperature, exclusively adopts a hairpin structure. However, under the same experimental conditions (5 mM DNA, no added salt, 295 K) d(CGCTAGCG) mainly adopts a B‐DNA‐type duplex. At lower temperatures (290 K) the hairpin form of d(CGaCTAGCG) occurs in slow exchange with an intact B‐DNA‐type duplex. When the DNA concentration of d(CGCTAGCG) is dramatically reduced (0.5 mM) the hairpin form becomes highly favoured at the expense of the dimer. Moreover, proton‐chemical‐shift considerations indicate that the structural features of the hairpin structure of d(CGCTAGCG) mimic, in part, those of the modified octamer d(CGaCTAGCG), i.e. a loop comprising only the two central residues with the thymine located into the minor groove (Pieters, J. M. L., de Vroom, E., van der Marel, G. A., van Boom, J. H., Koning, T. M. G., Kaptein, R. and Altona, C. unpublished results).

Thermodynamic analysis of d(CGCTAGCG) yields an average Tmd value of 342 K (1 M DNA) and a ΔHd° value of −266 kJ/mol for the dimer/coil transition and an average Tmh value of 321 K and ΔHh°−102 kJ/mol for the hairpin/coil equilibrium. For the duplex/coil equilibrium of d(CGaCTAGCG) an average Tmd value of 336 K (1 M DNA) and ΔHd° value of −253 kJ/mol are deduced. The hairpin/coil transition of d(CGaCTAGCG) is characterized by a ΔHh° value of −104 kJ/mol and an average Tmh value of 331 K. It is concluded that incorporation of an arabinofuranosylcytidine in the octamer d(CGaCTAGCG) results in stabilization of the hairpin form, whereas the dimer is destabilized by two aC · dG base pairs.
Synthesis of different nucleoside 5′‐diphospho‐sulfoquinovoses and their use for studies on sulfolipid biosynthesis in chloroplasts
Authors: HEINZ Ernst , SCHMIDT Hermann , HOCH Monika , JUNG Karl‐Heinz , BINDER Harald , SCHMIDT Richard R.
Published: March 2005

6‐Sulfo‐α‐D‐quinovopyranosyl phosphate was reacted with different nucleoside monophosphate morpholidates to form ADP‐, CDP‐, GDP‐ and UDP‐sulfoquinovose. Analytical and preparative HPLC of these nucleotides was performed on reversed‐phase columns using volatile buffer systems as eluant. The isolated compounds were characterized by NMR spectroscopy (except the CDP derivative) and used for an investigation of sulfolipid biosynthesis by chloroplasts. For this purpose intact spinach chloroplasts were biosynthetically preloaded with radioactive diacylglycerol to provide a sulfoquinovosyl acceptor. When sulfosugar nucleotides were added to such prelabelled intact organelles, the background levels of sulfolipid biosynthesis did not rise. On the other hand, after osmotic shock of prelabelled chloroplasts sulfolipid labelling was significantly increased by the addition of UDP‐ or GDP‐sulfoquinovose. The same stimulation was observed with isolated envelope membranes, and UDP‐sulfoquinovose proved to be twice as active as the GDP derivative. From these results it was concluded that the final step in sulfolipid biosynthesis is catalyzed by a UDP‐sulfoquinovose: 1,2‐diacylglycerol 3‐O‐α‐D‐sulfoquinovosyltransferase. This chloroplast enzyme cannot use exogenously supplied sulfosugar nucleotides, which as membrane‐impermeable compounds are expected to be formed in vivo within chloroplasts.
Glucosephosphate isomerase from Trypanosoma brucei
Authors: MARCHAND Martine , KOOYSTRA Ulco , WIERENGA Rik K. , LAMBEIR Anne‐Marie , Van BEEUMEN Jos , OPPERDOES Fred R. , MICHELS Paul A. M.
Published: March 2005

In Trypanosoma brucei the enzyme glucose‐6‐phosphate isomerase, like most other enzymes of the glycolytic pathway, resides in a microbody‐like organelle, the glycosome. Here we report a detailed study of this enzyme, involving a determination of its kinetic properties and the cloning and sequence analysis of its gene. The gene codes for a polypeptide of 606 amino acids, with a calculated Mr of 67280. The protein predicted from the gene sequence has 54–58% positional identity with its yeast and mammalian counterparts. Compared to those other glucose‐6‐phosphate isomerases the trypanosomal enzyme contains an additional 38–49 amino acids in its N‐terminal domain, as well as a number of small insertions and deletions. The additional amino acids are responsible for the 5‐kDa‐larger subunit mass of the T. brucei enzyme, as measured by gel electrophoresis. The glucose‐6‐phosphate isomerase of the trypanosome has no excess of positive residues and, consequently, no high isoelectric point, in contrast to the other glycolytic enzymes that are present in the glycosome. However, similar to other glycosomal proteins analyzed so far, specific clusters of positive residues can be recognized in the primary structure.

Comparison of the kinetic properties of the T. brucei glucose‐6‐phosphate isomerase with those of the yeast and rabbit muscle enzymes did not reveal major differences. The three enzymes have very similar pH profiles. The affinity for the substrate fructose 6‐phosphate (Km= 0.122 mM) and the inhibition constant for the competitive inhibitor gluconate 6‐phosphate (Ki= 0.14 mM) are in the same range as those of the similar enzymes. The Km shows the same strong dependence on salt as the rabbit muscle enzyme, although somewhat less than the yeast glucose‐6‐phosphate isomerase. The trypanocidal drug suramin inhibits the T. brucei and yeast enzymes to the same extent (Ki= 0.29 and 0.36 mM, respectively), but it had no effect on the rabbit muscle enzyme. Agaricic acid, a potent inhibitor of various glycosomal enzymes of T. brucei, has also a strong, irreversible effect on glucose‐6‐phosphate isomerase, while leaving the yeast and mammalian enzymes relatively unaffected.
Isolation of similar rolipram‐inhibitable cyclic‐AMP‐specific phosphodiesterases from rat brain and heart
Authors: NÉMOZ Georges , MOUEQQIT Mohammed , BRIGENT Annie‐France , PACHECO Henri
Published: March 2005

A cyclic AMP phosphodiesterase form of rat brain cytosol was purified by means of affinity chromatography on an immobilized analog of the specific inhibitor rolipram, followed by an exclusion chromatography step. The resulting preparation presented two protein bands in polyacrylamide gel electrophoresis, both with phosphodiesterase activity. Kinetics of cyclic AMP hydrolysis by the purified enzyme proved of the Michaelis type, with a Km of 3 μM, while hydrolysis of cyclic GMP displayed anomalous, negatively cooperative kinetics. At micromolar concentrations, this enzyme form hydrolyzed highly specifically cyclic AMP (50‐fold faster than cyclic GMP). Cyclic GMP proved a poor competitor of cyclic AMP hydrolysis (Ki 1.04 mM). The neurotropic compound, rolipram, strongly inhibited the enzyme, in a competitive manner (Ki 0.9 μM). This enzyme displayed a molecular mass of around 44 kDa as determined by exclusion chromatography, but two molecular masses of 42 kDa and 89 kDa were observable by electrophoresis on a polyacrylamide gradient gel, compatible with an equilibrium between dimeric and monomeric forms. Isoelectric focusing of the preparation gave rise to two activity peaks of pI 4.8 and 6.7, with identical properties. probably representing two charge isomers of the same protein. An enzyme prepared from rat heart cytosol by the same techniques as for brain phosphodiesterase isolation shared numerous characteristics with the enzyme of cerebral origin, suggesting identity of the rolipramsensitive form between the two tissues.

Since the rolipram‐sensitive form detected in crude brain preparations markedly differs from the above‐described isolated enzyme, both by its molecular mass in exclusion chromatography and by its pI, it is suggested that an alteration of the native protein, due to dissociation of putative subunits, occurs during the purification procedure.
Two types of cytochrome cd1 in the aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114
Authors: DOI Michio , SHIOI Yuzo , MORITA Masakazu , TAKAMIYA Ken‐içhiro
Published: March 2005

Components I and II of cytochrome cd1 which had different spectral features were purified from the aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114. Component I showed an absorption maxima at 700 and 406 nm in the oxidized form, and at 621, 552.5, 548 and 416 nm in the reduced form. Component II showed an absorption maxima at 635 and 410 nm in the oxidized form and at 628, 552,5, 548 and 417 nm in the reduced form. The relative molecular mass, Mr, of both cytochromes was determined to be 135000 with two identical subunits. Components I and II showed pI values of 7.6 and 6.8, respectively. The redox potential of hemes ranged form +234 mV to +242 mV, except for the heme d1 of component I (Em7=+ 134 mV). Components I and II showed both cytochrome c oxidase and nitrite reductase activities. Cytochrome c oxidase activity was strongly inhibited by a low concentration of nitrite and cyanide. Erythrobacter cytochromes c‐551 and c‐552 were utilized as electron donors for the cytochrome c oxidase reaction. The high affinity of cytochrome c‐552 to component II (Km= 1.27 μM) suggested a physiological significance for this cytochrome. Erythrobacter cytochromes cd1 are unique in their presence in cells grown under aerobic conditions as compared to other bacterial cytochromes cd1 which are formed only under denitrifying conditions.
Studies of the cellulolytic system of Trichoderma reesei QM 9414
Authors: VAN TILBEURGH Herman , LOONTIENS Frank G. , ENGELBORGS Yves , CLAEYSSENS Marc
Published: March 2005

Binding onto cellobiohydrolase II from Trichoderma reesei of glucose, cellobiose, cellotriose, derivatized and analogous compounds, is monitored by protein‐difference‐absorption spectroscopy and by titration of ligand fluorescence, either at equilibrium or by the stopped‐flow technique. The data complete earlier results [van Tilbeurgh, H., Pettersson, L. G., Bhikhabhai, R., De Boeck, H. and Claeyssens, M. (1985) Eur. J. Biochem. 148, 329–334] indicating an extended active center, with putative subsites ABCD.

Subsite A specifically complexes with β‐d‐glucosides and d‐glucose; at 25°C the latter influences the concomitant binding of other ligands at neighbouring sites. For several ligands this cooperative effect for binding (at 0.33 M glucose and temperature range 4–37°C) was characterized by a substantial increase of the enthalpic term (ΔΔH°=–35 kJ mol–1). Glucose (0.33 M) decreases the association and dissociation rate parameters of 4‐methylumbelliferyl β‐d‐cellobioside by one order of magnitude: k+= (3.6 ± 0.5)×10–5 M–1 s–1 versus (5.1 ± 0.1)×10–6 M–1 s–1 (in the absence of glucose) and k−= (1.3 ± 0.1) s−1 versus (14.0 ± 0.3) s–1.

As deduced from substrate‐specificity studies and inhibition experiments, subsite B interacts with terminal non‐reducing glucopyranosyl residues of oligomeric ligands and substrates, whereas catalytic (hydrolytic) cleavage occurs between C and D.

Association constants 10–100 times higher than those for cellobiose or its glycosides were observed for d‐glucopyranosyl‐(1 → 4)‐β‐d‐xylopyranose and cellobionolactone derivatives, suggesting ‘transition‐state’‐type binding for these ligands at subsite C.

Although subsite D can accomodate a bulky chromophoric group (MeUmb) its preference for a glucosyl residue is reflected in the lower binding enthalpy of cellotriose (–34 kJ mol–1) as compared to cellobiose (–28.3 kJ mol–1) and MeUmb(Glc)2 (–11.6 kJ mol–1).

This model indicates that oligomeric ligands (substrates) interact through cooperativity of their subunits at the extended binding site of cellobiohydrolase II.
Marker enzymes in rat liver vesicles involved in transcellular transport
Authors: QUINTART Joël , BAUDHUIN Pierre , COURTOY Pierre J.
Published: March 2005

In order to label the vesicles involved in transcellular transfer (transcytosis) through hepatocytes, polymeric IgA (pIgA) was conjugated to horseradish peroxidase (HRP) and injected into rats. The endosomes containing this ligand at 10 or 20 min after injection were isolated by the diaminobenzidine‐induced density‐shift procedure and their content in various marker enzymes was measured. The endosomes carrying pIgA‐HRP 10 min after injection contained only traces of 5′‐nucleotidase and low amounts of alkaline phosphodiesterase I. The estimated marker enzyme content is similar to that observed for the particles containing galactosylated bovine serum albumin conjugated to HRP, a ligand degraded in lysosomes. However, 20 min after injection, the transcytotic endosomes showed a marked enrichment in 5′‐nucleotidase and especially in alkaline phosphodiesterase I. The results confirm the heterogeneity of rat liver endosomes and substantiate the concept of distinct endosomal compartments.
Catalytic activity and denaturation of enzymes in water/organic cosolvent mixtures
Authors: MOZHAEV Vadim V. , KHMELNITSKY Yuri L. , SERGEEVA Mariya V. , BELOVA Alla B. , KLYACHKO Nataliya L. , LEVASHOV Andrey V. , MARTINEK Karel
Published: March 2005

The dependence of the catalytic activities of α‐chymotrypsin and laccase on the concentration of organic cosolvents (alcohols, glycols and formamides) in mixed aqueous media has a pronounced threshold character: it does not change up to a critical concentration of the non‐aqueous cosolvents added, yet further increase of the latter (by only a small percentage, by vol.) leads to an abrupt decrease in enzyme activity. Fluorescence studies indicate that the inactivation results from reversible conformational changes (denaturation) of the enzymes. There is a linear correlation between the critical concentration of residual water (at which the enzyme inactivation occurs in a threshold manner) and the hydrophobicity of the organic cosolvents added. A quantitative criterion is suggested for the selection of organic cosolvents to be used for enzymatic reactions in homogeneous water/organic solvent media.
Site‐directed mutagenesis of the spinach acyl carrier protein‐I prosthetic group attachment site
Authors: JAWORSKI Jan G. , POST‐BEITTENMILLER Martha A. , OHLROGGE John B.
Published: March 2005

Site‐directed mutagenesis was used to change the phosphopantetheine attachment site (Ser38) of spinach acyl carrier protein I (ACP‐I) from a serine to a threonine or cysteine residue.

Although the native ACP‐I is fully phosphopantethenylated when expressed in Escherichia coli, the THACP‐I and CY‐ACP‐I mutants were found to be completely devoid of the phosphopantetheine group. Therefore, the E. coli holoACP synthase requires serine for in vivo phosphopantetheine addition to spinach ACP‐I.

Spinach holoACP‐synthase was completely inactive in vitro with either the TH‐ACP‐I or CY‐ACP‐I mutants. In addition, TH‐ACP‐I and CY‐ACP‐I were strong inhibitors of spinach holoACP synthase.

The mutant ACPs were weak or ineffective as inhibitors of spinach fatty acid synthesis and spinach oleoyl‐ACP hydrolase.

Compared to holoACP‐I, the mutant apoACP‐I analogs had: (a) altered mobility in SDS and native gel electrophoresis, (b) altered binding to anti‐(spinach ACP‐I) antibodies and (c) altered isoelectric points. The combined physical, immunological and enzyme inhibition data indicate that attachment of the phosphopantheine prosthetic group alters ACP conformation.
Protein transfer from an aqueous phase into reversed micelles
Authors: WOLBERT Ronnie B. G. , HILHORST Riet , VOSKUILEN Gijsbert , NACHTEGAAL Henk , DEKKER Matthijs , RIET Klaas VAN'T , BIJSTERBOSCH Bert H.
Published: March 2005

Proteins are spontaneously transferred from an aqueous solution into reversed micelles, provided the aqueous phase has the proper composition. Besides the composition of the aqueous phase, the composition of the organic phase and the properties of the proteins also play a role. We studied uptake profiles of 19 proteins as a function of pH of the aqueous solution. The organic phase consisted of trioctylmethylammonium chloride and nonylphenol pentaethoxylate (Rewopal HV5) as surfactant, octanol as cosurfactant and isooctane as continuous phase. In all cases, except for rubredoxin, proteins were transferred at pH values above their isoelectric point. The pH where maximal solubilization takes place can be described by the relationship: pHoptimum= isoelectric point +0.11 × 10–3Mr–0.97. So, the larger the protein, the more charge is needed to provide the energy required for the adaptation of the micellar size to the protein size. For protein transfer into sodium di‐(2‐ethylhexyl)sulphosuccinate (AOT) reversed micelles a similar relationship was found. The percentage of protein transferred could be related to the symmetry of charge distribution over the protein. This symmetry was expressed as the % of random electric moments on a protein that is larger than the effective electric moment of the protein (% S) [Barlow, D. J. and Thornton, J. M. (1986) Biopolymers 25, 1717]. The larger the value of % S, the more homogeneously the charges are distributed and the lower the percentage transfer.
Acyl carrier protein is present in the mitochondria of plants and eucaryotic micro‐organisms
Authors: CHUMAN Lorraine , BRODY Stuart
Published: March 2005

Proteins antigenically similar to the acyl carrier protein (ACP) found in the mitochondria of Neurospora crassa were detected by immunoblotting and radioimmunoassay techniques in mitochondria isolated from yeast, potatoes, and pea leaves. These mitochondrial proteins were similar to Neurospora ACP both in their electrophoretic mobility and in their unusual decrease in mobility upon reduction. Authentic ACP(s) show this type of change upon conversion of the acylated to the unacylated form. Purified ACP from both spinach chloroplasts and Escherichia coli cells cross‐reacted with antibodies raised against Neurosopora ACP. Purified ACP from, Neurospora cross‐reacted with antibodies raised against spinach chloroplast ACP and E. coli ACP. Mitochondria isolated from beef heart and rat brain were tested extensively and exhibited no cross‐reaction with any of the three anti‐ACP preparations. The discovery of ACP in the mitochondria of other organisms raises questions concerning the possible relationship between ACP and β‐oxidation in mitochondria, the involvement of ACP in de novo biosynthesis of some of the acyl chains in mitochondria and the subcellular locations of fatty acid biosynthesis in plants and eucaryotic micro‐organisms.
The metabolic consequences of hydroperoxide perfusion on the isolated rat heart
Authors: CHATHAM John C. , GILBERT Hiram F. , RADDA George K.
Published: March 2005

Perfusion of rat hearts with Krebs‐Henseleit bicarbonate buffer containing low concentrations of hydrogen peroxide or t‐butylhydroperoxide (50–500 μM) caused an imbalance in the relative synthesis versus utilization rates of ATP, leading to a net hydrolysis of ATP and phosphocreatine. Hydrogen peroxide also caused an 80% inactivation of glyceraldheyde‐3‐phosphate dehydrogenase, resulting in an inhibition of glycolysis and a rapid accumulation of sugar phosphates as detected with 31P‐NMR spectroscopy. This inhibition was partially reversible with peroxide‐free perfusion, resulting in a cessation of high‐energy‐phosphate hydrolysis and a decrease in the accumulated inorganic phosphate and sugar phosphate. t‐Butylhydroperoxide toxicity was irreversible. Providing an alternative, non‐glycolytic substrate (butyrate) did not protect against the toxicity of hydrogen peroxide, but altered the relative importance of sugar phosphate formation versus ATP hydrolysis. Experiments with heart homogenates in vitro suggest that the inhibition of glyceraldehyde‐3‐phosphate dehydrogenase is a consequence of a direct reaction of the enzyme with hydrogen peroxide or one of its metabolites. Hearts subjected to total global ischemia (10–20 min), followed by reperfusion with oxygenated buffer, showed no detectable inactivation of glyceraldehyde‐3‐phosphate dehydrogenase, indicating that ischemia and reperfusion do not result in the production of high global concentrations of hydrogen peroxide.
Degradation of tomato pathogenesis‐related proteins by an endogenous 37‐kDa aspartyl endoproteinase
Authors: RODRIGO Ismael , VERA Pablo , CONEJERO Vicente
Published: March 2005

As a response to the stress induced by different afflicting agents, tomato plants (Lycopersicon esculentum) produce the so‐called ‘pathogenesis‐related’ proteins. Here we report the degradation of some of these proteins by a constitutive endoproteolytic activity that co‐distributes with pathogenesis‐related proteins in the intercellular spaces of tomato leaves infected with citrus exocortis viroid. This endoproteinase was purified, showing a pH optimum of 2.5–3.5, a Mr of 37000 and selective inhibition by pepstatin. In crude homogenates, the enzyme does not seem to degrade other cellular proteins. This specificity indicates that the proteinase might be involved in the extracellular degradative pathway of pathogenesis‐related proteins and in the regulation of their biological function.
Differential exposure of aromatic amino acids in the red‐light‐absorbing and far‐red‐light‐absorbing forms of 124‐kDa oat phytochrome
Authors: SINGH Bal Ram , SONG Pill‐Soon , EILFELD Peter , RÜDIGER Wolfhart
Published: March 2005

The surface topography of aromatic amino acid residues and/or other hydrophobic groups of phytochrome has been investigated by ultraviolet absorption spectra and ultraviolet circular dichorism using phytochromecyclodextrin inclusion complexation. Three different types of cyclodextrins (α, β and γ) with varying hydrophobic cavity sizes, were used. Complexation resulted in significant changes in the circular dichroic signals of both the red‐light‐absorbing (Pr) and far‐red‐light‐absorbing (Pfr) forms of phytochrome in the ultraviolet region at 222 nm, mid‐ultraviolet at 280 nm and 300 nm and in the near‐ultraviolet and visible regions at 365 nm and 670 nm, respectively. α‐ and β‐Cyclodextrins were markedly (1.7–4.5‐fold) more effective in reducing the midultraviolet CD signal of Pr than that of Pfr, indicating a differential inclusion of the aromatic amino acid residues. γ‐Cyclodextrin did not exhibit any significant differentiation. Secondary structure analysis of the phytochrome‐cyclodextrin complexes revealed a considerable increase in the α‐helical contents of both Pr and Pfr forms. The increase in the Pfr form (17–25%) was about twice that in the Pr form (8–9%), indicating a differential effect of complexation on the conformation of the phytochrome protein. Although the photostationary‐state equilibrium of the phytochrome was not affected by the cyclodextrin complexation, the Pr → Pfr phototransformation rate was significantly increased. However, the Pfr → Pr photoreversion was not affected significantly. The results suggest a differential complexation of cyclodextrins with the Pr and Pfr forms of phytochrome as a result of a difference in accessibility of aromatic amino acids in the two forms. A detailed analysis of absorption difference spectra and circular dichroic spectra around 280 nm also revealed evidence for a difference in the exposure of aromatic amino acids.
Fatty‐acid‐binding protein from bovine brain
Authors: SCHOENTGEN Françoise , PIGNÈDE Georges , BONANNO Laurent Michel , JOLLÈS Pierre
Published: March 2005

A fatty‐acid‐binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G‐75 filtration and electrofocusing. The purified FABP behaved as an anionic protein with an apparent molecular mass of 14.7 kDa; its complete amino acid sequence was determined and microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP and bovine mammary‐derived growth inhibitor (MDGI).
Purification of a vasodilator‐regulated phosphoprotein from human platelets
Authors: HALBRÜGGE Maria , WALTER Ulrich
Published: March 2005

Cyclic‐nucleotide‐elevating vasodilators such as prostaglandin E1, prostacyclin, sodium nitroprusside and endothelium‐derived relaxing factor inhibit both contraction of vascular smooth muscle cells and the aggregation of platelets at an early step of the activation cascade.

Previous studies from this laboratory [Waldmann, R., Nieberding, M. and Walter, U. (1987) Eur. J. Biochem. 167, 441–448) established that in human platelets cyclic‐nucleotide‐elevating vasodilators stimulated a pattern of protein phosphorylation which was mediated by both cAMP‐ and cGMP‐dependent protein kinases. Of particular interest was a membrane‐bound 50‐kDa protein whose phosphorylation was increased both by cAMP‐and cGMP‐elevating vasodilators in intact platelets and by endogenous cAMP‐ and cGMP‐dependent protein kinase in platelet membranes.

Since the molecular mechanism of action of cyclic‐nucleotide‐elevating vasodilators is unknown, this 50‐kDa phosphoprotein from human platelets was purified to apparent homogeneity by salt extraction, anion, cation and dye‐ligand chromatography. The purified protein migrated as a 46‐kDa protein in SDS/PAGE, was an excellent substrate for both cAMP‐ and cGMP‐dependent protein kinases and migrated in SDS/PAGE as a 50‐kDa protein after phosphorylation by these protein kinases. Analysis by limited proteolysis, tryptic fingerprinting and of phosphoamino acids established that the purified protein is identical with the 50‐kDa protein phosphorylated by both cAMP‐ and cGMP‐dependent protein kinases in platelet membranes and in response to cAMP‐ and cGMP‐elevating vasodilators with intact platelets. Evidence is presented that the purified protein contains at least two phosphorylation sites, each of which is preferentially phosphorylated by either cAMP‐ or cGMP‐dependent protein kinase. The availability of this vasodilator‐regulated phosphoprotein as a purified protein should now allow new approaches for investigating the function of this protein and its possible role in the mechanism of action of cyclic‐nucleotide‐elevating vasodilators.
Slow/cardiac sarcoplasmic reticulum Ca2+‐ATPase and phospholamban mRNAs are expressed in chronically stimulated rabbit fast‐twitch muscle
Authors: LEBERER Ekkehard , HÄRTNER Karl‐Thomas , BRANDL Christopher J. , FUJII Junichi , TADA Michihiko , MACLENNAN David H. , PETTE Dirk
Published: March 2005

Fast‐twitch extensor digitorum longus muscles of the rabbit were subjected to chronic low‐frequency stimulation during different time periods. Changes in the relative amounts of mRNAs encoding fast and slow/cardiac Ca2+ ‐ATPase isoforms were assessed through the use of an R Nase‐protection assay. Stimulation‐induced increases in slow/cardiac Ca2+ ‐ATPase and phospholamban mRNAs were quantified by mRNA hybridization. Prolonged stimulation resulted in an exchange of the fast with the slow/cardiac Ca2+ ‐ATPase isoform mRNAs. The exchange was complete after 72 d of stimulation as compared with normal slow‐twitch soleus muscle. The tissue content of phospholamban mRNA reached levels similar to that found in normal slow‐twitch soleus muscle by the same time. The conversion of the sarcoplasmic reticulum coincided with the fast‐to‐slow troponin C isoform transition, previously investigated in the same muscles.
Molecular cloning and sequence analysis of the cDNA for rat mitochondrial enoyl‐CoA hydratase
Authors: MINAMI‐ISHII Naoko , TAKETANI Shigeru , OSUMI Takashi , HASHIMOTO Takashi
Published: March 2005

To elucidate structural relationships between the mitochondrial and peroxisomal isozymes of β‐oxidation systems, cDNA of the mitochondrial enoyl‐CoA hydratase was cloned and sequenced. The 1454‐bp cDNA sequence contained a 870 bp of open reading frame, encoding a polypeptide of 290 amino acid residues. When compared with the amino‐terminal sequence of the mature enzyme, the predicted sequence contained a 29‐residue presequence at the amino terminus. This presequence had characteristics typical of a mitochondrial signal peptide. The primary structure of this enzyme showed significant similarity with the amino‐terminal portion of sequence of the peroxisomal enoyl‐CoA hydratase: 3‐hydroxyacyl‐CoA dehydrogenase bifunctional enzyme. The carboxy‐terminal part of the latter enzyme has sequence similarity with mitochondrial 3‐hydroxyacyl‐CoA dehydrogenase [Ishii, N., Hijikata, M., Osumi, T. & Hashimoto, T. (1987) J. Biol. Chem. 262, 8144–8150]. These findings suggest that the peroxisomal bifunctional enzyme has the hydratase and dehydrogenase functions on the amino‐ and carboxy‐terminal sides, respectively. The mitochondrial β‐oxidation enzymes and the peroxisomal bifunctional enzyme may have common evolutionary origins.
Scalar, dipolar‐correlated and J‐resolved 2D‐NMR spectroscopy of the specific phenolic mycoside of Mycobacterium tuberculosis
Authors: DAFFÉ Mamadou , SERVIN Philippe
Published: March 2005

Two‐dimensional chemical shift correlated (COSY), nuclear Overhauser (NOESY) and J‐resolved spectroscopy were used to determine the complete structure of the major triglycosyl dimycocerosyl phenol phthiocerol of the tubercle bacillus (strain Canetti) without any other analytical technique. The COSY spectrum of the native glycolipid allowed the composition of the trisaccharide and the location of one methoxyl group to be determined through the assignment of the resonances of the non‐anomeric methine protons. Information with respect to the configuration of the sugar residues, the pyranose structure and the linkage sites of the trisaccharide were obtained by the analysis of the COSY spectrum of the peracetylated glycolipid. The NOESY spectrum confirmed the linkage sites through the inter‐residue connectivity across glycosidic linkages and allowed the determination of the sequence of the oligosaccharide and the configuration of its sugar residues from the through‐space connectivities. The J‐resolved spectroscopy was used to elucidate the structure of the glycolipid and gave additional data showing the presence of 11 non‐equivalent methyl groups and allowing the recognition of two other analogues of the phenol phthiocerol derivative in the mixture. Thus, the non‐destructive 2D‐NMR spectroscopy was found to be satisfactory for the analysis of mycobacterial mycosides.
Tryptophan 110, a residue involved in the toxic activity but not in the enzymatic activity of notexin
Authors: MOLLIER Pascale , CHWETZOFF Serge , BOUET Françoise , HARVEY Alan L. , MÉNEZ André
Published: March 2005

We prepared two derivatives of notexin, a phospholipase A2 from Notechis scutatus scutatus venom, by modifying the protein with 2‐nitrophenylsulfenylchloride, a tryptophan‐specific reagent. One derivative was modified at both tryptophans 20 and 110 whereas the other was modified at tryptophan 20. Evidence based on circular dichroic analysis and antigenicity towards a notexin‐specific monoclonal antibody indicated that derivatization at both tryptophans did not affect the tertiary structure of notexin. Concomitant modification of tryptophans 20 and 110 induced a marked decrease in the capacity of notexin to kill mice and to block neuromuscular transmission in the chick biventer cervicis preparation, whereas selective modification at tryptophan 20 had no effect on the lethal properties of notexin. This implies that the decrease in the lethal properties of notexin after derivatization was due to modification at tryptophan 110. However, the diderivatized notexin retained full enzymatic activity, implying that neither tryptophan 20 and tryptophan 110 are involved in the catalytic function of the molecule. We conclude that notexin harbours two functional sites. One of them corresponds to the enzymatic site, whereas the other, which includes tryptophan 110, provides specific toxic characteristics to notexin. By reference to previous crystallographic studies, the relative spatial positions of elements involved in toxicity and the catalytic site, we propose a possible orientation of notexin with respect to its putative membrane‐bound target.