bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of proteolytic fragments of the laminin‐nidogen complex and their activity in ligand‐binding assays
Authors: MANN Karlheinz , DEUTZMANN Rainer , TIMPL Rupert
Published: March 2005

Some 12 new nidogen and laminin fragments were purified from elastase, thrombin and trypsin digests and characterized by their sizes (22 kDa to > 300 kDa), subunit patterns on electrophoresis, partial amino acid sequences, content of specific epitopes and their binding to laminin or nidogen structures in radioligand assays. This permitted the various fragments to be ordered along the dumbbell‐shaped structure of nidogen and to compare them with previously described nidogen fragments arising by endogenous proteolysis. Two nidogen fragments (E‐50, E‐90; 50 kDa and 90 kDa) remain associated with a large laminin fragment in elastase digests of the complex and could be dissociated with 2 M guanidine · HCl. Recombination studies demonstrated Kd= 10–20 nM for this interaction. Nidogen fragments devoid of binding activity included the tryptic peptide T‐40 (40 kDa) corresponding to the rod‐like domain and several larger fragments extending more to the N‐terminus of nidogen. An N‐terminal thrombin fragment of about 50 kDa was also inactive. Together the data show a lack of laminin binding to the N‐terminal globule and rod of nidogen and provide indirect evidence that this activity is located within or close to its C‐terminal globular domain.

Nidogen‐binding structures of laminin were obtained as two large fragments (> 300 kDa), P1X and E1X. They correspond to the short arm structure of laminin with one (E1X) or two (P1X) arms decreased in size to the inner rod‐like segment. Shortening in E1X is mainly due to the B1 chain segment including the central globular domain which was identified as a new laminin fragment E10. Binding of E1X and P1X to nidogen was comparable to that of laminin while much lower activity was found for other laminin fragments. A 10‐fold lower binding potential was also observed for the laminin‐nidogen complex whose structure can now be defined in more precise molecular terms.
Alternative mechanisms for gene activation induced by poly(rI) · poly(rC) and Newcastle disease virus
Authors: LAMMERS Reiner , GROSS Gerhard , MAYR Ulrich , COLLINS John
Published: March 2005

After poly(rI) · poly(rC) induction of FS‐4 fibroblasts, both human interferon‐β (IFN‐β) mRNA and an additional induced RNA class (12S RNA) hybridize to a genomic cosmid clone containing the human IFN‐β gene as well as 35 kbp of flanking sequences. However, this coinduced 12S RNA does not originate from regions in the neighborhood of the IFN‐β gene, but hybridizes to the genomic cosmid clone via repetitive Alu‐family sequences. While IFN‐β mRNA rapidly decays after reaching a maximum 2–4 h after induction, this 12S RNA is stably maintained in the fibroblast cell for more than 16 h. Contrary to IFN‐β mRNA, the level of the 12S RNA is not further elevated by superinduction conditions (cycloheximide treatment) during poly(rI) · poly(rC) induction. However, subsequent to treatment with the weaker viral inducer Newcastle disease virus (NDV) both IFN‐β and the 12S RNA transcripts are induced to a higher level in the presence of cycloheximide. Cell‐free translation of hybrid‐selected 12S RNA leads to detection of an induced protein of 14 kDa. cDNA cloning reveals that the 12S RNA contains part of an Alu‐family sequence in the 5′‐untranslated region. The 12S RNA is probably not an RNA polymerase III transcript and codes for a protein of 9 kDa (as monitored by in vitro cell‐free translation). This discrepancy in molecular mass can be attributed to a retarded migration of the protein in SDS/PAGE.
The production of normal and variant human glucose‐6‐phosphate dehydrogenase in cos cells
Authors: MASON Philip J. , VULLIAMY Tom J. , FOULKES Nicholas S. , TOWN Margaret , HAIDAR Buran , LUZZATTO Lucio
Published: March 2005

Full‐lenght cDNA coding for human glucose‐6‐phosphate dehydrogenase (G6PD) was inserted into a eukaryotic expression vector containing the immediate early promotor of cytomegalovirus. When this plasmid was introduced into cos cells by transfection it led to the production of high levels of human G6PD. cDNAs containing mutations found in G6PD‐deficient individuals were constructed by in vitro mutagenesis and expressed in the same system. Characterization of the G6PD proteins obtained in this way confirmed the primary structure inferred for the variant enzymes. An enzyme in which lysine‐205 had been mutated to threonine was produced and found to have no G6PD activity, proving that this lysine residue is essential for enzyme activity in human G6PD.
Alterations in the d(CpGpT) structure in solution as a result of [PtCl(diethylenetriamine)]+ binding
Authors: van GARDEREN Carla J. , ALTONA Cornelis , REEDIJK Jan
Published: March 2005

The trinucleotide d(CpGpT) reacts with [PtCl(dien)]Cl (dien = diethylenetriamine) to yield as a single adduct Pt(dien)[d(CpGpT)‐N7(2)]. The structure of this adduct in solution has been analysed with the aid of NMR spectroscopy and compared with that of the unmodified trinucleotide. A change in the population of the S conformer of the guanosine deoxyribose ring and a syn preference of the guanine residue are the most important changes occurring upon platination. As a result the dC‐dG stack disappears, whereas the dG‐dT stack is hardly affected. The CD spectra of both platinated and free d(CpGpT) confirm the different nature of the two molecules.
Inhibition of uridine phosphorylase by pyrimidine nucleoside analogs and consideration of substrate binding to the enzyme based on solution conformation as seen by NMR spectroscopy
Authors: VERES Zsuzsa , NESZMÉLYI András , SZABOLCS Anna , DÉNES Géza
Published: March 2005

Some 3′‐ and/or 5′‐substituted pyrimidine nucleosides, as well as anhydropyrimidine nucleosides, which have no flexibility about the N‐glycosidic bond were studied as inhibitors of thymidine phosphorylase and uridine phosphorylase. The conformation of some analogs was also investigated in order to obtain information on substrate binding to the enzyme.

The above compounds, including the potential anti‐(human immunodeficiency virus) agent, 3′‐azido‐2′,3′‐dideoxy‐5‐methyluridine were not substrates for either thymidine phosphorylase or uridine phosphorylase. (The only exception was arabinofuranosyl‐5‐ethyluracil, which proved to be a poor substrate for uridine phosphorylase).

The phosphorolysis of thymidine by thymidine phosphorylase was slightly or not at all altered by these pyrimidine nucloside analogs. The lowest Ki was obtained in the case of 3′‐azido‐2′,3′‐dideoxy‐5‐methyluridine and the highest in the case of 2′‐deoxylyxofuranosyl‐5‐ethyluracil, when studying the analogs with flexible structure as inhibitors of uridine phosphorylase. The Ki for 2,3′‐ and 2,5′‐anhydro‐2′‐deoxy‐5‐ethyluridine was 5–6 orders of magnitude higher than that for 2,2′‐anhydro‐5‐ethyluridine. Competitive inhibition was observed in all cases.

For these three molecules computer‐aided molecular modelling predicts the following glycosidic torsion angles X(O4,‐C1,‐N1‐C2): 109° for 2,2′‐anhydro‐5‐ethyluridine, and 78° and 71° for 2,3′‐ and 2,5′‐anhydro‐2′‐deoxy‐5‐ethyluridine respectively. These values are corroborated by high‐resolution 13C‐ and 1H‐NMR studies.

2′‐Deoxy‐5‐ethyluridine is predicted to have a syn conformation with X= 46° and ΔE about 2.5 kJ/mol over the minimum energy (in anti position, X=−147°). 1H and 13C data including homonuclear Overhauser enhancements complete the information about the solution conformation.

Considering the Ki values obtained, it is likely that substrates of uridine phosphorylase will bind to the enzyme in the same conformation as 2,2′‐anhydro‐5‐ethyluridine. The >30° deviation from the N‐glycosidic torsion angle of 2,2′‐anhydro‐5‐ethyluridine results in much higher Ki values.
Accumulation of carbamoylphosphate‐synthetase and phosphoenolpyruvate‐carboxykinase mRNA in embryonic rat hepatocytes
Authors: van ROON Marian A. , ZONNEVELD Duco , CHARLES Robert , LAMERS Wouter H.
Published: March 2005

The aim of this study was to establish whether the initial accumulation of hepatocyte‐specific proteins after hormone induction is regulated at the pretranslational and/or the translational level. To this end, mRNA molar concentrations were determined and compared with rates of protein synthesis from previous studies [van Roon, M. A., Charles, R. & Lamers, W. H. (1987Eur. J. Biochem. 165, 229–234].

In vivo, carbamoylphosphate‐synthetase mRNA starts to accumulate at day 17 of pregnancy. Phosphoenol‐pyruvate‐carboxykinase mRNA starts to accumulate only just prior to birth. Embryonic day 14 (i.e. 8 days before the expected day of birth), livers were chosen to study the regulation of the initiation of hepatocyte‐specific mRNA accumulation in vitro. Accumulation of carbamoylphosphate‐synthetase and phosphoenolpyruvate‐carboxykinase mRNA is regulated by the same hormones as accumulation of the respective proteins. The rate at which carbamoylphosphate‐synthetase and phosphoenolpyruvate‐carboxykinase mRNA molecules accumulate in cultured embryonic, hepatocytes is relatively low, compared to that of postnatal hepatocytes. However, the increase of the rate of synthesis of carbamoylphosphate‐synthetase and phosphoenolpyruvate‐carboxykinase protein is even 3‐6‐fold slower than that of mRNA. This shows that initially mRNAs accumulate intracellularly to a relatively high concentration without being efficiently translated or translatable. Only after the mRNA concentration reaches a plateau at 72 h and 48 h respectively, the cellular capacity to synthesize the respective proteins increases. Therefore, the translational efficiency is certainly one of the major rate‐limiting factors of the initial phases of expression of the hepatocyte‐specific genes for carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase.
Large‐scale preparation and reconstitution of apo‐flavoproteins with special reference to butyryl‐CoA dehydrogenase from Megasphaera elsdenii
Authors: VAN BERKEL Willem J. H. , VAN DEN BERG Willy A. M. , MÜLLER Franz
Published: March 2005

A new method is described for the large‐scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic‐interaction chromatography.

Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl‐CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability.

The holoenzymes are bound to phenyl‐Sepharose CL‐4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5–4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200–500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol.

The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo‐lipoamide‐dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at ‐18°C.

Steady‐state fluorescence‐polarization measurements of protein‐bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme‐FAD complex.

The holoenzyme of butyryl‐CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50–80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at ‐18°C.

From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl‐CoA dehydrogenase is governed by both the pH‐dependent hydrodynamic properties of apoenzyme and the pH‐dependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native‐like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence‐polarization measurements.

The degree of reconstitution of butyryl‐CoA dehydrogenase is more than 80% of the original specific activity under certain conditions. Unliganded reconstituted enzyme is easily regreened in the presence of CoASH and Na2S.

The large‐scale preparation and reconstitution of the apoproteins of glutathione reductase from human erythrocytes and mercuric reductase from Pseudomonas aeruginosa is also described.
Structural determination of the photosystem II core complex from spinach
Authors: IRRGANG Klaus‐D. , BOEKEMA Egbert J. , VATER Joachim , RENGER Gernot
Published: March 2005

A photosystem II core complex was purified with high yield from spinach by solubilization with β‐dodecylmaltoside. The complex consisted of polypeptides with molecular mass 47, 43, 34, 31, 9 and 4 kDa and some minor components, as detected by silver‐staining of polyacrylamide gels. There was no indication for the chlorophyll‐a/b‐binding, light‐harvesting complex polypeptides. The core complex revealed electron‐transfer activity (1,5‐diphenylcarbazide → 2,6‐dichloroindophenol) of about 30 μmol reduced 2,6‐dichloroindophenol/mg chlorophyll/h. The structural integrity was analyzed by electron microscopy. The detergent‐solubilized protein complex has the shape of a triangular disk with a maximum diameter of 13 nm and a maximum height of 6.8 nm. The shape of this core complex differs considerably from that of cyanobacterial photosystem II membrane fragments, which are elongated particles. The structural differences between both the complexes of higher plants and cyanobacteria are discussed with special emphasis on their association with the antenna apparatus in the photosynthetic membranes.
Probing the active site of flavocytochrome b2 by site‐directed mutagenesis
Authors: REID Graeme A. , WHITE Scott , BLACK Michael T. , LEDERER Florence , MATHEWS F. Scott , CHAPMAN Stephen K.
Published: March 2005

The three‐dimensional structure of flavocytochrome b2 (L‐lactate dehydrogenase) from bakers' yeast (Saccharomyces cerevisiae) has recently been solved at 0.24‐nm resolution [Mathews & Xia (1987) in Flavins and flavoproteins, Walter de Gruyter, Berlin, pp. 123–131]. We have used this structural information to investigate the roles of particular amino acid residues likely to be involved in the oxidation of L‐lactate by kinetic analysis of mutant enzymes generated by site‐directed mutagenesis of the isolated gene.

The hydroxyl group of Tyr254 was expected to be important for the abstraction of the hydroxyl proton of L‐lactate in the oxidation to pyruvate. Replacement of this tyrosine by phenylalanine reduced kcat from 190 ± 3 s−1 (25°C, pH 7.5) to 4.3 ± 0.1 s−1. This substitution had, however, no discernable effect on Km for lactate (0.54 ± 0.03 mM for the mutant compared with 0.49 ± 0.03 mM for the wild‐type enzyme). Arg376 was expected to be essential for productive binding and orientation of L‐lactate. Replacing Arg376 with lysine abolished all detectable activity. A total loss of enzymic activity was also observed when Lys349, thought likely to stabilize the anionic form of the flavin hydroquinone, was replaced by arginine. An amino acid residue replacement at a distance from the active site, Ala306 to serine, had a minor but significant effect on kcat (reduced from 190 s−1 to 160 s−1) and Km (increased from 0.49 mM to 0.83 mM) presumably arising from small conformational effects. The implications of these results are discussed in relation to the mechanism of l‐lactate oxidation.
Interaction of Escherichia coli translation‐initiation factor IF‐1 with ribosomes
Authors: CELANO Bruna , PAWLIK Roman T. , GUALERZI Claudio O.
Published: March 2005

The interaction between Escherichia coli translation‐initiation factor IF‐1 and ribosomes was studied in binding experiments by Airfuge centrifugation. IF‐1 binds to the 30S, but not to the 50S, ribosomal subunit and its binding is strongly stimulated by IF‐3 and IF‐2, either alone or in combination. From the dependence of the Kd of the 30S‐subunit –IF‐1 complex on ionic strength, it can be concluded that IF‐1 binds primarily via an ionic interaction, most likely with the 16S rRNA, with the minimum number of ion pairs involved being 2.7–3.6. The 30S‐ subunit–IF‐1 interaction is unaffected by temperature changes between 11°C and 44°C and is thus accompanied by a negligible enthalpy change. It is concluded that the interaction is an entropy‐driven process triggered mainly by the release of counter ions from the RNA phosphates.

Titration of 30S‐subunit–IF‐1 complexes with 50S subunits causes the ejection of the factor indicating that IF‐1 is released from the ribosomes during the subunit association step which marks the transition from a 30S‐initiation‐complex to a 70S initiation complex.
Kinetic and biochemical properties of CTP:choline‐phosphate cytidylyltransferase from the rat brain
Authors: MAGES Francoise , REY Catherine , FONLUPT Pierre , PACHECO Henri
Published: March 2005

In order to investigate the mechanisms involved in some brain disorders at the membrane level, we studied the kinetics and biochemical properties of brain CTP:choline‐phosphate cytidylyltransferase (EC 2.7.7.15), the rate‐limiting enzyme of the two‐step biosynthesis of phosphatidylcholine. This enzyme catalyzes the biosynthesis of CDPcholine from choline phosphate and CTP. We found that its subcellular localization (mainly in microsomal and cytosolic fractions) was different from that of phosphatidylethanolamine N‐methyltransferase (EC 2.1.1.17), the enzyme of the alternative pathway for phosphatidylcholine synthesis.

CTP:choline‐phosphate cytidylyltransferase showed a Km of 10 mM for CTP and 0.3 mM for choline phosphate and exhibited a random mechanism. CDPcholine, the reaction product, was a competitive inhibitor of choline phosphate and CTP utilization and had a Ki of 0.090 mM. Both particulate and soluble enzymes required Mg2+ and exhibited an optimal pH at about 7.

Cytosolic activity was enhanced by addition of unsaturated fatty acids or phospholipids extracted from brain membranes. Such an enhancement was increased with the centrifugation time used for preparing the soluble enzyme.
Thyroid‐specific and cAMP‐dependent hypersensitive regions in thyroglobulin gene chromatin
Authors: HANSEN Carole , GERARD Catherine , VASSART Gilbert , STORDEUR Patrick , CHRISTOPHE Daniel
Published: March 2005

Two regions hypersensitive to DNase I digestion were found in a 7‐kb segment of thyroglobulin gene 5′‐flanking sequences in the chromatin from bovine thyroid.

The most upstream site (−2000 to −1600 bp relative to the transcriptional start) was found in thyroid chromatin only, but independently of actual expression of the gene. It therefore represents a tissue‐specific characteristic which may be associated with the commitment of the thyroglobulin gene to transcriptional activity.

The very 5′ end of the gene and the proximal promoter sequences (−100 to +60 bp relative to transcriptional start), constitute the second site, the hypersensitive character of which could be directly correlated with transcriptional activity. The structural changes occurring in this region of the chromatin were dependent on cAMP stimulation of the thyroid cells.
Isolation and characterization of the bovine k‐casein gene
Authors: ALEXANDER Leeson J. , STEWART A. Francis , MACKINLAY Antony G. , KAPELINSKAYA Tatiana V. , TKACH Tatiana M. , GORODETSKY Stanislav I.
Published: March 2005

The bovine k‐casein gene has been isolated as a series of overlapping λ clones and shown to consist of five exons distributed over a total length of approximately 13 kb. Most of the mature protein‐coding sequence is contained in a single large exon.

Approximately 65% of the gene has been sequenced together with portions of the 5′‐ and 3′‐flanking sequences. The immediate 5′‐flanking sequence contains several motifs which are characteristic of upstream regions including a TATA box, a CAAT box, a sequence similar to that recognized by transcription factor AP‐1 and a purine‐rich sequence resembling that found upstream in all other lactoprotein genes. Other possible regulatory sequences are found upstream of exon 4.

The organization of the k‐casein gene, together with its upstream sequence, confirms previous conclusions that it is unrelated to the calcium‐sensitive‐casein gene familiy to which it is linked. Evidence is presented which supports a previous suggestion that k‐casein and the fibrinogens are evolutionarily related.

Intron sequences contain several examples of the A family of the artiodactyl Alu‐like repeated sequences, together with a single example of a C‐family sequence. The remainders of the introns of the k‐casein gene, compared with the repeat elements and exons, are A + T‐rich.

Among the Δ clones isolated, representatives were found of the A and B genetic variants which can be distinguished by restriction‐enzyme analysis. Several other examples of polymorphisms in the non‐coding region were found.
Complete amino acid sequence of the neurone‐specific γ isozyme of enolase (NSE) from human brain and comparison with the non‐neuronal α form (NNE)
Authors: McALEESE Sybil M. , DUNBAR Bryan , FOTHERGILL John E. , HINKS Lesley J. , DAY Ian N.M.
Published: March 2005

The complete amino acid sequence (433 residues) of the human neurone‐specific γ isozyme of enolase (NSE) has been determined by a combination of direct amino acid sequencing and nucleotide sequencing of cloned cDNA. Substantial amino acid sequence of the non‐neuronal α form of the enzyme was also obtained which agreed almost entirely with the indirect cDNA sequence [9]. Comparison of the two human sequences shows no insertions or deletions, but 72 replacements. Comparison of the human γ form with the corresponding isozyme from the rat shows only 7 replacements (compared to 27 changes between the human and rat α isozymes). We have identified regions of sequence difference between the human α and γ forms that are mainly hydrophilic in character (residues 271–285, 298–316 and 416–433). These residues are on the surface of the three‐dimensional structure [21] and could be useful as immunogens to produce antibodies specific for the neurone‐specific form.
Catalytic‐site mapping of pyruvate formate lyase
Authors: PLAGA Wulf , FRANK Rainer , KNAPPE Joachim
Published: March 2005

Pyruvate formate‐lyase of Escherichia coli cells, a homodimeric protein of 2 × 85 kDa, is distinguished by the property of containing a stable organic free radical (g= 2.0037) in its resting state. The enzyme (E‐SH) achieves pyruvate conversion to acetyl‐CoA via two distinct half‐reactions (E‐SH + pyruvate ⇌ E‐S‐acetyl + formate; E‐S‐acetyl + CoA ⇌ E‐SH + acetyl‐CoA), the first of which has been proposed to involve reversible homolytic carbon‐carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332–1335].

Present studies identified Cys‐419 as the covalent‐catalytic cysteinyl residue via CNBr fragmentation of E‐S‐ [14C]acetyl and radio‐sequencing of the isolated peptide CB‐Ac (amino acid residues 406–423). Reaction of the formate analogue hypophosphite with E‐S‐acetyl was investigated and found to produce 1‐hydroxyethyl‐phosphonate with a thioester linkage to the adjacent Cys‐418. The structure was determined from the chymotryptic peptide CH‐P (amino acid residues 415–425), using 31P‐NMR spectroscopy (δ= 44 ppm) and by chemical characterisation through degradation into 1‐hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P‐C‐bond synthesis involves the enzyme‐based free radical and is proposed to resemble the physiological C–C‐bond synthesis (pyruvate production) from formate and E‐S‐acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate‐lyase.

The central Cys‐418/Cys‐419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.
Visualization of reversible macromolecular reactions in an analytical ultracentrifuge
Authors: CHERNYAK Victor Ya. , KOGAN‐YASNY Victor V.
Published: March 2005

It has been shown that the saw‐like disturbances of sedimentation observed in an analytical ultracentrifuge are not caused by convective disturbances of the solution but result from a special type of intermolecular reaction of reversible association/dissociation. A qualitative theory of saw‐like anomalies has been suggested and the sedimentation and kinetic conditions of their origin have been indicated. Such reactions are a frequent occurrence in the serum of patients affected with rheumatic diseases and acute myocardial infarction. Experimental data indicate the involvement of immunoglobulins (viz., low‐affinity antibodies) which form reversible immune complexes. Saw‐like sedimentation patterns, especially those of the schlieren type, are a direct testimony to reversible association/dissociation reactions in macromolecular solutions, whereas other experimental methods provide only oblique evidence.
Electron‐transport‐driven sodium extrusion during methanogenesis from formaldehyde and molecular hydrogen by Methanosarcina barkeri
Authors: MÜLLER Volker , WINNER Christiane , GOTTSCHALK Gerhard
Published: March 2005

Methanogenesis from formaldehyde or formaldehyde + H2, as carried out by Methanosarcina barkeri, was strictly dependent on sodium ions whereas methane formation from methanol + H2 or methanol + formaldehyde was Na+‐independent. This indicates that the reduction of formaldehyde to the formal redox level of methanol exhibits a Na+ requirement. During methanogenesis from formaldehyde, a ΔpNa in the range of ‐62 mV to ‐80 mV was generated by means of a primary, electron‐transport‐driven sodium pump. This could be concluded from the following results obtained on cell suspensions of M. barkeri.

The addition of proton conductors or inhibitors of the Na+/H+ antiporter had no effect on sodium extrusion.

During methanogenesis from formaldehyde + H2 a ΔΨ of ‐60 mV to ‐70 mV was generated even in the presence of proton conductors.

ATPase inhibitors, applied in the presence of proton conductors, had no effect on primary sodium extrusion or generation of a ΔΨ. Evidence for a Na+‐translocating ATPase could not be obtained.
Site‐specific phosphorylation of the purified receptor for calcium‐channel blockers by cAMP‐ and cGMP‐dependent protein kinases, protein kinase C, calmodulin‐dependent protein kinase II and casein kinase II
Authors: JAHN Holger , NASTAINCZYK Wolfgang , RöHRKASTEN Axel , SCHNEIDER Toni , HOFMANN Franz
Published: March 2005

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium‐channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165‐kDa and the 55‐kDa proteins of the purified CaCB receptor. The 130/28‐kDa and the 32‐kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165‐kDa subunit with 2–3‐fold higher initial rate than the 55‐kDa subunit. Casein kinase II phosphorylated the 165‐kDa and the 55‐kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55‐kDa protein. The 55‐kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two‐dimensional peptide maps of the 165‐kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55‐kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165‐kDa and the 55‐kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.
Isoleucyl‐tRNA synthetase from baker's yeast
Authors: FREIST Wolfgang , STERNBACH Hans
Published: March 2005

Specificity with regard to amino acids in aminoacylation of tRNAIle‐C‐C‐3′dA by isoleucyl‐tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values. The lowest values are observed for Cys, Val, His, and Trp (D2= 180–1700), indicating that at same amino acid concentrations isoleucine is 180–1700 times more attached to tRNAIle‐C‐C‐3′dA. The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2= 10 000–30 000). D2 values of the other amino acids are in the range of 2000–10 000.

Recognition of most amino acids is achieved in a four‐step process. Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof‐reading steps occur on the pre‐ and the post‐transfer stage. For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post‐transfer proof‐reading is negligible. As a special case in discrimination of valine, one initial discrimination step and the post‐transfer proof‐reading step are lacking.

The role of the terminal hydroxyl groups of the tRNA for post‐transfer proof‐reading is assigned to a simple neighbouring group effect. No preference for the 2′ or 3′ position in proof‐reading can be postulated.
cDNA cloning and complete primary structure of the small, active subunit of human carboxypeptidase N (kininase 1)
Authors: GEBHARD Wolfgang , SCHUBE Matthias , EULITZ Manfred
Published: March 2005

The human plasma metallo‐protease carboxypeptidase N of Mr 280 000 consists of two small, enzymatically active subunits of Mr 50 000 and two large subunits. Only the large subunits are glycosylated. They may have a function in stabilizing the complex in plasma. The N‐terminal sequence of the small subunit was determined from the isolated protein and used to specify a unique 59‐mer oligonucleotide probe. A cDNA clone of 1.7 kbp containing the entire coding sequence of the small subunit of carboxypeptidase N was isolated from a human‐liver cDNA library. The cDNA clone encodes a signal sequence of 20 amino acids and the 438 aminc acids of the mature subunit. There is a remarkable primary structure similarity of 49% to bovine carboxypeptidase E (enkephalin convertase). A more distant relationship to the bovine pancreatic, digestive carboxypeptidases A and B or even to the metallo‐endopeptidases is based mainly on the occurrence of conserved, mechanistically important residues.
Purification of GTP cyclohydrolase I from human liver and production of specific monoclonal antibodies
Authors: SCHOEDON Gabriele , REDWEIK Udo , CURTIUS Hans‐Christoph
Published: March 2005

GTP cyclohydrolase I, the first enzyme in the de novo biosynthesis of tetrahydrobiopterin, was enriched more than 13000‐fold from human liver by preparative isoelectric focusing using Sephadex G‐200 SF gels. The pI of the active enzyme was determined as 5.6 by analytical isoelectric focusing in the same matrix. The native enzyme has an apparent molecular mass of 440 kDa and appears to be composed of eight 50‐kDa subunits as estimated from SDS/PAGE. The enriched enzyme preparation was used to produce specific monoclonal antibodies. From 11 monoclonal antibodies obtained, one was extensively characterized for further applications. This monoclonal antibody belongs to the IgM class and shows immunoreactivity with GTP cyclohydrolase I both from man and from Escherichia coli. It is capable of highly sensitive detection of GTP cyclohydrolase I by ELISA and by Western blot analysis. The monoclonal antibody was used for the immunoenzymatic localisation of GTP cyclohydrolase I in human peripheral blood mononuclear cells. Furthermore, it was possible to demonstrate the absence of immunoreactivity in cells with GTP cyclohydrolase I deficiency. The antibody's use as a tool either for differential diagnosis of atypical phenylketonuria due to GTP cyclohydrolase I deficiency or prenatal diagnosis of this severe inherited metabolic disease is now under investigation.
An atypical heme‐binding structure of cytochrome c1 of Euglena gracilis mitochondrial complex III
Authors: MUKAI Kuniaki , YOSHIDA Masaki , TOYOSAKI Hiroshi , YAO Yoshio , WAKABAYASHI Sadao , MATSUBARA Hiroshi
Published: March 2005

Complex III was purified from submitochondrial particles prepared from Euglena gracilis. The purified complex consisted of 10 subunits and lost antimycin sensitivity. The Euglena complex III showed an atypical difference absorption spectrum for cytochrome c1 with its α‐band maximum at 561 nm. The pyridine ferrohemochrome prepared from covalently bound heme in the Euglena complex III had an α‐peak at 553 nm. This wavelength is the same as that of pyridine ferrohemochrome prepared from Euglena mitochondrial cytochrome c (c‐558), the heme of which is linked to only a single cysteine residue through a thioether bond. Cytochrome c1 which was a heme‐stained subunit with a molecular mass of 32.5 kDa was isolated from the purified complex III and its N‐terminal sequence of 46 amino acids was determined. On the basis of apparent homologies to cytochromes c1 from other sources, this sequence included the heme‐binding region. However, the amino acid at position 36, corresponding to the first cysteine involved in heme linkage in other cytochromes c1, was phenylalanine. Position 39, corresponding to the second cysteine, was not identified despite the treatment for removal of the heme and carboxymethylation of the expected cysteine. The unidentified amino acid is assumed to be a derivative of cysteine to which the heme is linked through a single thioether bond. The histidine‐40 corresponding to the probable fifth ligand for heme iron was conserved in Euglena cytochrome c1.
The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis
Authors: PAUL Wyatt , MERRICK Mike
Published: March 2005

Active Fe protein of nitrogenase was synthesised in a non‐nitrogen fixing organism when Escherichia coli was transformed with a plasmid encoding only two nif‐specific genes, nifH and nifM of Klebsiella pneumoniae. Hence proteins NifH and NifM are sufficient to produce active Fe protein in E. coli.

K. pneumoniae strains carrying chromosomal nifW− and nifZ− mutations were constructed and shown to be significant C2H2‐reducing activity and to grow on N‐free plates. Nevertheless, derepressing cultures of the mutant strains had reduced levels of MoFe protein activity, and consequently significantly lower levels of nitrogenase activity, than the nif+ parent strain. NifW and NifZ therefore appear to be involved in the formation or accumulation of active MoFe protein, but are not essential for nitrogen fixation in K. pneumoniae under the conditions tested.
A 19F‐NMR study of 2‐fluoro‐4‐demethoxydaunomycin intercalation complexes with the decanucleotides d(G‐C)5 and d(A‐T)5
Authors: HAMMER Brendon C. , RUSSELL Richard A. , WARRENER Ronald N. , COLLINS J. Grant
Published: March 2005

Binding configurations and equilibria of intercalation complexes formed by the novel anthracycline drug, 2‐fluoro‐4‐demethoxydaunomycin (2FD), with the decanucleotides d(G‐C)5 and d(A‐T)5 have been studied by 19F‐NMR spectroscopy.

The 19F chemical shift of 2FD bound to d(A‐T)5 was ∼ 1.5 ppm downfield of that observed for 2FD bound to d(G‐C)5. By mixing equimolar amounts of aqueous d(G‐C)5, d(A‐T)5 and 2FD, the distribution of drug between the nucleotides was followed by observing relative peak intensities and showed no G‐C or A‐T binding preference at room temperature.

It was shown that each decanucleotide duplex bound three 2FD molecules, giving a neighbour exclusion parameter, n, of n= 3 for this drug. The stoichiometric complexes, which we denote by [d(A‐T)5][2FD]3 and [d(G‐C)5][2FD]3, were also purified and isolated in this study.
Photochemically‐induced dynamic nuclear polarization proton‐NMR of aequorin discharged by calcium‐independent light emission
Authors: RAY Bruce D. , SCHEEK Ruud M. , KEMPLE Marvin D. , PRENDERGAST Franklyn G. , NAGESWARA RAO B. D.
Published: March 2005

Photochemically‐induced dynamic nuclear polarization was used to identify exposed amino‐acid residues and to assign resonances in the 1H‐NMR spectrum of Ca(II)‐independent discharged (inactivated) aequorin. A previous nuclear magnetic resonance, circular dichroism and fluorescence study [Ray, B. D., Ho, S., Kemple, M. D., Prendergast, F. G. and Nageswara Rao, B. D. (1985) Biochemistry 24, 4280–4287] indicated that as the Ca(II)‐activated bioluminescent protein aequorin from jellyfish spontaneously emits light in the absence of Ca(II), it changes from a rigid, fully active form to a discharged form in which a number of amino‐acid residues are more mobile than in the native protein. Laser‐photochemically‐induced dynamic nuclear polarization experiments identified tryptophan and tyrosine residues, but not histidine residues, in Ca(II)‐independent discharged aequorin to be accessible to the flavin dye used. These exposed residues are also among the mobile residues of the Ca(II)‐independent discharged protein. Resonances of all the protons (including the α protons) of the accessible tryptophan and tyrosine residues were assigned with the aid of two‐dimensional photochemically‐induced dynamic nuclear polarization J‐correlated spectroscopy. The oxidized chromophore, from which light is emitted in aequorin, was not accessible to the dye in the Ca(II)‐independent discharged protein. No exposed residue was detected in the photochemically‐induced dynamic nuclear polarization spectrum of Ca(II)‐independent discharged aequorin from which the oxidized chromophore was removed, corroborating the previous finding that in this apo‐discharged form the protein partially refolds and thereby loses some of the mobility acquired in the formation of the Ca(II)‐independent discharged protein.
Conformational analysis of the sialylα(2→3/6)N‐acetyllactosamine structural element occurring in glycoproteins, by two‐dimensional NOE 1H‐NMR spectroscopy in combination with energy calculations by hard‐sphere exo‐anomeric and molecular mechanics force‐field with hydrogen‐bonding potential
Authors: BREG Jan , KROON‐BATENBURG Loes M. J. , STRECKER Gérard , MONTREUIL Jean , VLIEGENTHART Johannes F. G.
Published: March 2005

The conformation is described of the sialylα(2 → 3/6)N‐acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAcα(2 → 3) Galβ(1 → 4)GlcNAcβ(1 → N)Asn and NeuAcα‐(2 → 6)Galβ(1 → 4)GlcNAcβ(1 → N)Asn is presented and for each glycosidic linkage, except for the α(2→6)‐linkage, a number of interglycosidic NOEs are measured. The analysis of these effects is performed using a full relaxation matrix. Analysis of intraresidue NOEs provides a calibration of the calculation method. Hard‐sphere exo‐anomeric (HSEA) energy calculations indicate a single conformation for the β(1 → 4)‐linkage in both compounds, both being consistent with the NOE data. HSEA and molecular‐mechanics force‐field with hydrogen‐bonding potential energy calculations both indicate the existence of three preferred conformations for the α(2 → 3)‐linkage. The analysis of the NOE spectra are consistent with a distribution over two or three of these conformations; by combination with the energy diagram for this linkage the existence of onyl a single conformation can be excluded. The NOE spectrum of the compound with the α(2 → 6)‐linkage indicates a gt orientation for the Gal C‐6 hydroxymethyl group. On this basis, the HSEA energy calculations for the α(2→6)‐linkage indicate an extended low‐energy surface with a number of preferred conformations. The absence of NOEs across this linkage is interpreted in terms of a non‐rigid, but overall folded conformation of the NeuAcα(2 → 6)Galβ(1 → 4)GlcNAcβ structural element. This provides an explanation for the shift effects induced by α(2 → 6) attachment of NeuAc to the N‐acetyllactosamine unit.
Interactions between discrete neuronal membrane binding sites for the putative K+‐channel ligands β‐bungarotoxin, dendrotoxin and mast‐cell‐degranulating peptide
Authors: BREEZE Alexander L. , DOLLY J. Oliver
Published: March 2005

β‐Bungarotoxin, a presynaptically active neurotoxin from the venom of Bungarus multicinctus, was radiolabelled with 125I and its binding to synaptic membranes from rat brain was analyzed. The interaction of these binding sites with those for dendrotoxin (a convulsant polypeptide from mamba venom) and mast‐cell‐degranulating peptide (from bee venom) was examined in the light of the known effects of all three toxins on voltage‐dependent K+ currents.

When measured in Krebs/phosphate buffer, the binding appeared monotonic at low concentrations of radioiodinated β‐bungarotoxin (Kd 0.4 nM; Bmax 0.42 pmol/mg protein); higher concentrations of labelled toxin revealed an additional binding component of lower affinity, but computer analysis of the data failed to provide well‐defined estimates of its Kd and Bmax values.

Equilibrium binding experiments conducted in imidazole‐based buffers yielded distinctly biphasic Scatchard plots; computer analysis of the data revealed two populations of sites [Kd 0.26 (±0.30) nM and 6.14 (±5.68) nM; Bmax 0.16 (± 0.20) and 2.65 (± 1.21) pmol/mg protein].

In Krebs medium, β‐bungarotoxin was a very weak antagonist of the binding of 125I‐labelled dendrotoxin. In imidazole medium, however, the efficacy of the inhibition was markedly increased; analysis of this inhibition showed it to be non‐competitive.

Dendrotoxin inhibited the binding of radioiodinated β‐bungarotoxin in Krebs medium with high potency, although the interaction was by a complex, non‐competitive mechanism.

Mast‐cell‐degranulating peptide inhibited non‐competitively the binding of both radiolabelled dendrotoxin and β‐bungarotoxin but with relatively low potency.

A speculative schematic model of the dendrotoxin β‐bungarotoxin/mast‐cell‐degranulating peptide binding component(s) is proposed. Findings are discussed in terms of the likely involvement of these sites with voltagedependent K+‐channel proteins.
Developmental regulation of expression of the regulatory subunit of the cAMP‐dependent protein kinase of Blastocladiella emersonii
Authors: MARQUES Marilis do Valle , JULIANI Maria Helena , MAIA José Carlos C. , GOMES Suely Lopes
Published: March 2005

A monospecific polyclonal antiserum to the regulatory subunit (R) of the cAMP‐dependent protein kinase of Blastocladiella emerosonii has been developed by immunization with purified regulatory subunit. In Western blots, the antiserum displays high affinity and specificity for the intact R monomer of Mr= 58000, as well as for its proteolytic products of Mr= 43000 and Mr= 36000, even though the antiserum has been raised against the Mr= 43000 fragment. Western blots of cell extracts prepared at different times during the life cycle of the fungus indicate that the increase in cAMP‐binding activity occurring during sporulation, as well as its decrease during gremination, are associated with the accumulation of the regulatory subunit during sporulation and its disappearance during germination, respectively. Pulse labeling with [35S]methionine and immunoprecipitation indicate that the accumulation of R is due to its increased synthesis during sporualtion. Two‐dimensional gel electrophoresis of affinity purified cell extracts obtained after [35S]methionine pulse labeling during sporualtion confirms de novo synthesis of R during this stage and furthermore shows that the protein is rapidly phosphorylated after its synthesis. In vitro translation studies using RNA isolated from different stages of the life cycle following by immumoprecipitation have shown that the time course of expression of the mRNA coding for the regulatory subunit parallels the rate of its synthesis in vivo.
Structural features of lipoprotein lipase
Authors: PERSSON Bengt , BENGTSSON‐OLIVECRONA Gunilla , ENERBÄCK Sven , OLIVECRONA Thomas , JÖRNVALL Hans
Published: March 2005

A structural homology between lipoprotein lipase, pancreatic lipase and hepatic lipase is known and indicates that all three lipases are members of a common protein family. Lipoprotein lipase and pancreatic lipase utilize small protein co‐factors, apolipoprotein C‐II and co‐lipase, respectively, but comparisons reveal no homology between the co‐factor molecules. Hence, they do not show the same relationship as their target enzymes. Neither do screenings detect any extensive similarities between lipoprotein lipase, serine hydrolases, or apolipoproteins. Scannings against data bank proteins show that a 105‐residue segment of lipoprotein lipase exhibits a 35–40% residue identity with a sub‐region of Drosophila vitellogenins. One fifth of the conserved amino acid residues (8 of 40) are glycine, a pattern which is typical of distantly related forms of protein families. This supports a true relationship between large segments of Drosophila vitellogenins and lipases. Physiological and functinal aspects of the vitellogenin/lipoprotein lipase similarities are given. The region concerned is entirely within the N‐terminal domain of lipoprotein lipase and constitutes the segment where the similarity to hepatic and pancreatic lipases is most pronounced. Within this lipase region a 10‐residue putative lipid‐binding site exists for which further similarities have been found to the otherwise not closely related lingual/gastric lipases, prokaryotic lipases and lecithin‐cholesterol acyltransferase. Another segment in lipoprotein lipase, where the heparin‐binding site has been mapped, exhibits a correlation between strength of heparin binding and extent of basic residues among members of the lipase family. It further exhibits weak similarities with the ‘Zn‐finger’ DNA‐binding segment of steroid hormone receptors and may indicate convergence in a binding interaction. Thus, a functional subdivision of lipoprotein lipase into different segments can be distinguished.
Evidence indicating that the glucagon‐induced increase in cytoplasmic free Ca2+ concentration in hepatocytes is mediated by an increase in cyclic AMP concentration
Authors: STADDON James M. , HANSFORD Richard G.
Published: March 2005

The mechanism whereby glucagon causes an increase in the concentration of cytoplasmic free Ca2+, [Ca2+]c, in isolated hepatocytes has been investigated. There have been proposals of cyclic‐AMP‐dependent and cyclic‐AMP‐independent mechanisms. In this work, the inactivation of pyruvate kinase was used as an indicator of increases in the activity of cyclic‐AMP‐dependent protein kinase, A‐kinase. [Ca2+]c was measured using the fluorescent probe indo‐1. The decrease in activity of pyruvate kinase caused by an increase in [Ca2+]c alone, i. e. mediated by mechanisms not involving cyclic AMP and exemplified by the effect of vasopressin, was of minimal significance under the conditions of the enzyme assay. Studies of the effects of a wide range of glucagon concentrations indicate that any increase in [Ca2+]c caused by glucagon was always associated with a decrease in pyruvate kinase activity. A similar relationship was obtained if glucagon‐receptor occupancy was circumvented by using the 8‐bromo‐derivative of cyclic AMP to activate the A‐kinase. It was also found that the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine could potentiate the ability of glucagon to increase [Ca2+]c: no such potentiation was observed when vasopressin was used to raise [Ca2+]c. Together these data indicate that an increase in cyclic AMP concentration, sufficiently great to activate A‐kinase, is a mechanism that mediates the glucagon‐induced increase in [Ca2+]c.