bluuebunny/crossref_metadata_2025 huggingface dataset:
NMR and electron‐paramagnetic‐resonance studies of a dihaem cytochrome from Pseudomonas stutzeri (ATCC 11607) (cytochrome c peroxidase)
Authors: VILLALAÍN Jose , MOURA Isabel , LIU Ming Cheh , PAYNE William J. , LeGALL Jean , XAVIER António V. , MOURA José J. G.
Published: March 2005

A dihaem cytochrome (Mr 37400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11607). The haem redox potentials are far apart: one of the haemsis completely ascorbate‐reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states.

In the oxidized state, the low‐temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low‐spin haem indicating a histidinyl‐methionyl coordination; (II) a low‐spin haem indicating a histidinyl‐histidinyl coordination; and (III) a minor high‐spin haem component. At room temperature, NMR and optical studies indicate the presence of high‐spin and low‐spin haems, suggesting that for one of the haems a high‐spin to low‐spin transition is observed when temperature is decreased.

In the half‐reduced state, the component I (high redox potential) of the EPR spectrum disappears arid induces a change in the g‐values and linewidth of component II; the high‐spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high‐spin ferric and a low‐spin (methionyl‐coordinated) ferrous state. The NMR data fully support the haem‐haem interaction probed by EPR.

In the reduced state, the NMR spectrum indicates that the low‐potential haem is high‐spin ferrous.
The use of two‐dimensional nuclear‐magnetic‐resonance spectroscopy and two‐dimensional difference spectra in the elucidation of the active center of Megasphaera elsdenii flavodoxin
Authors: MOONEN Chrit T. W. , SCHEEK Ruud M. , BOELENS Rolf , MÜLLER Franz
Published: March 2005

1H‐1H ‘through bond’ correlated (COSY) and 1H‐lH ‘through space’ (NOESY) two‐dimensional NMR techniques were applied to study the structure of Megasphaern elsdenii flavodoxin in the oxidized and reduced state. It is shown that two‐dimensional NOESY difference spectra between spectra of flavodoxin in the reduced and semiquinone state are sensitive to the active center of the fully reduced state. The sphere of the active center observed in the difference spectra can be varied easily by changing the relative amount of flavodoxin semiquinone in the second sample. The difference NOESY spectra simplified the analysis of the complex spectra. Resonances could be assigned to Ala‐56, Tyr‐89 and Trp‐91, which arc located in the direct vicinity of the protein‐bound flavin. The relative positions and side‐chain dihedral angles of these residues are compared for the two redox states. Ala‐56 and Tyr‐89 show identical relative positions and dihedral angles in the two redox states, although the rotational motion of Tyr‐89 is enhanced in the oxidized state. In both redox states Trp‐91 is immobilized and extremely close to the prosthetic group. However, a small displacement of Trp‐91 towards the (N(5) atom of the flavin occurs upon reduction. The results obtained for Trp‐91 are in excellent agreement with crystallographic results of the related flavodoxin from Clostridium MP. However, the latter studies showed a somewhat different position of the tyrosine residue compared with our results.
Depletion of blood plasma cytidine due to increased hepatocellular salvage in d‐galactosamine‐treated rats
Authors: HOLSTEGE Axcl , MANGLITZ Dagmar , GEROK Wolfgang
Published: March 2005

Pyrimidine nucleosides in blood plasma of rats were identified by different procedures, including chemical peak shift methods, before their quantification by reversed‐phase high‐performance liquid chromatography. The concentrations of uridine, cytidine, and deoxycytidine were 1.0 ± 0.2, 10.6 ± 1.9, and 33.4 ± 5.4 μmol/l, respectively.

Six hours after the administration of d‐galactosamine, the level of circulating cytidine was severely depressed to 25% of control values; uridine decreased to 54% while deoxycytidine remained unchanged. 24 h after the dose of the amino sugar, the levels of cytidine and uridine returned to control values in blood plasma.

Total acid‐soluble uridine, cytidine, guanosine, and adenosine was determined by reversed‐phase HPLC after treatment of the neutralized acid‐soluble supernatant of freeze‐clamped rat livers with phosphodiesterase and alkaline phosphatase. Six hours after its administration, d‐galactosamine induced a 2.2‐fold and a 1.6‐fold rise in total acid‐soluble uridine and cytidine, respectively. Co‐administration of N‐(phosphonoacetyl)‐l‐aspartate, an inhibitor of de novo pyrimidine sythesis, suppressed the increase in total acid‐soluble uridine observed after d‐galactosamine alone, but was without effect on the enhancement of total cytidine.

Three hours after d‐galactosamine and 15 min after [2–14C] cytidine, there was a rapid fall of the labeled nucleoside in blood plasma to 49% of control animals accompanied by a 2.8‐fold rise in the total radioactivity of rat liver homogenates.

From these results it can be concluded that the hepatocellular rise in total acid‐soluble cytidine after d‐galactosamine, in contrast to the increase in total acid‐soluble uridine, originates from the phosphorylation of blood plasma cytidine via the salvage pathway. The depletion of circulating cytidine in the presence of hepatocellular UTP deficiency points to the importance of the liver and the hepatic UTP level for the clearance of blood plasma cytidine.
Nucleotide sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase
Authors: SPENCER Margaret E. , DARLISON Mark G. , STEPHENS Paul E. , DUCKENFIELD Ian K. , GUEST John R.
Published: March 2005

The nucleotide sequence of the sucB gene, which encodes the dihydrolipoamide succinyltransferase component (E2o) of the 2‐oxoglutarate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain‐termination method. The results extend by 1440 base pairs the previously reported sequence of 3180 base pairs, containing the sucA gene. The sucB structural gene comprises 1209 base pairs (403 codons excluding the initiating AUG), and it is preceded by a 14‐base‐pair intercistronic region containing a good ribosomal binding site. The absence of a typical terminator sequence and the presence of an IS‐like sequence downstream of sucB suggest that there may be further gene(s) in the suc operon. The IS‐like sequence is homologous with other intercistronic sequences including that between the sdhB and suc A genes, the overall gene organisation being: sdhB‐IS‐sucAsucB‐IS‐. The patterns of codon usage indicate that sucB may be more strongly expressed than suc A, consistent with the disproportionate contents of their products in the oxoglutarate dehydrogenase complex.

The predicted amino acid composition and Mr (43 607) of the succinyltransferase component agree with previous studies on the purified protein. Comparison with the corresponding acetyltransferase component of the pyruvate dehydrogenase complex (E2p, aceFgene product) indicates that each contains two analogous domains, an amino‐terminal lipoyl domain linked to a carboxy‐terminal catalytic and subunit binding domain. The lipoyl domain of the acetyltransferase (E2p) comprises three tandemly repeated ∼ 100‐residue lipoyl binding regions containing two short (∼19 residues) internal repeats, whereas the lipoyl domain of the succinyltransferase (E2o) contains just one ∼ 100‐residue lipoyl binding region, with ∼27% homology to each of the three comparable regions in E2p, and no detectable internal repeats. The catalytic and subunit binding domains, each ∼300 residues, have an overall homology of 34 % and, consistent with their combination of analogous and specific functions, some regions are more homologous than others. Both sequences feature segments rich in proline and alanine. In E2p these occur at the carboxy‐terminal ends of each of the three lipoyl binding regions, there being a particularly extended sequence at the end of the third repeat, whereas in E2o the main proline‐alanine segment is found ∼50 residues into the subunit binding domain. It is suggested that these segments could form flexible hinge‐like regions which may account for the observed mobility in these subunits and be important in the mode of action of the bound lipoyl coenzyme. The results provide a basis for defining the structure‐function relationships of the two acyltransferases and they suggest that these components are closely related in evolution.
Preparation of monoclonal antibodies against glycoprotein IIIa of human platelets
Authors: MELERO Jose A. , GONZALEZ‐RODRIGUEZ Jose
Published: March 2005

Monoclonal antibodies against purified glycoprotein IIIa (GPIIIa) of human platelet membranes have been obtained. These antibodies, except one, are able to bind to intact platelets; the exception is M108/p98 antibody which recognizes a new epitope, unmasked after proteolysis of GPIIIa in vitro. Several antigenic areas can be delineated on the molecule, by testing the ability of different antibodies to compete in their simultaneous binding to GPIIIa. One of the monoclonal antibodies inhibits ADP‐induced platelet aggregation while others do not have an effect or induce agglutination of platelets independent of ADP. Conventional antiserum raised against purified GPIIIa also blocks the aggregation induced by ADP. These results favour the hypothesis that GPIIIa plays a direct role in the mechanism of platelet aggregation.
The efficiency of oxidative phosphorylation and the rapid control by thyroid hormone of nicotinamide nucleotide reduction and transhydrogenation in intact rat liver mitochondria
Authors: CORRIGALL Jane , TSELENTIS Basil S. , MOWBRAY John
Published: March 2005

In confirmation of previous work enhancement of the fluorescence emission of reduced nicotinamide nucleotides in intact rat liver mitochondria was found to depend on incubation conditions.

Under standard conditions the enhancement is constant at 4.8‐fold in states 3 and 4 and is not altered by thyroidectomy of the animal 6 weeks prior to experiment.

The ADP‐induced (state 4 → state 3 → state 4) fluorescence changes are significantly different in intact mitochondria from normal and hypothyroid animals and reflect the decreased rate and efficiency of oxidative phosphorylation after thyroidectomy.

Incubation of liver homogenates in vitro for 15 min with 1 μM triiodothyronine before isolating mitochondria significantly restores their ADP response towards normal. Direct addition of hormone to isolated mitochondria was ineffective.

Enzymatic measurement of mitochondrial extracts shows that thyroidectomy leads to increases in the contents of NAD(H) by 22% and NADP(H) by 33%.

With glutamate as substrate ADP‐induced changes in the reduced/oxidized ratio of NAD+ are not significantly altered in hypothyroid preparations. By contrast the NADP+ ratio remains substantially more reduced in state 3 than it does in normal mitochondria.

The hypothesis is advanced that the decreased efficiency of hypothyroid preparations in phosphorylating ADP may be the result of increased energy‐linked transhydrogenase activity. This is needed to supply NADPH via the glutathione peroxidase for reducing endogenously formed peroxides.

Direct reduction of mitochondrial glutathione with dithiothreitol had no substantial effect on ADP/O ratios or on ADP‐induced redox cycles in either normal or thyroidectomised preparations. This decisively eliminates the possibility that lowered phosphorylation efficiency is the result of a leak of reducing equivalents via glutathione peroxidase.
Tissue distribution of messenger RNAs coding for opioid peptide precursors and related RNA
Authors: JINGAMI Hisato , NAKANISHI Shigetada , IMURA Hiroo , NUMA Shosaku
Published: March 2005

All of the endogenous opioid peptides thus far identified are derived from three types of precursors, i.e. the corticotropin/β lipotropin precursor, preproenkephalin A and preproenkephalin B. Poly(A)‐containing RNA from various bovine and porcine tissues has been subjected to blot hybridization analysis with the use of cDNA probes specific for the three opioid peptide precursors. Analysis with a corticotropin/β lipotropin precursor cDNA probe has revealed, in addition to the pituitary mRNA, a smaller hybridizable RNA species present in bovine extrapituitary tissues, such as the adrenal medulla, thyroid, thymus, duodenum and lung. The hypothalamus contains both these RNA species. DNA complementary to the smaller RNA species from the bovine adrenal medulla has been cloned. Analysis of the cloned cDNA, in conjunction with endonuclease S1 mapping of poly(A)‐rich RNA from the adrenal medulla, has indicated that the smaller RNA species represents the 3′‐terminal 712–729 nucleotides, excluding the poly(A) tail, of the pituitary corticotropin/β‐lipotropin precursor mRNA, having heterogeneous start sites. Analysis with a preproenkephalin A cDNA probe has shown the presence of hybridizable RNA in the bovine hypothalamus, duodenum and pituitary neurointermediate lobe in addition to the adrenal medulla. The hybridizable RNA species from all these tissues are indistinguishable in size. RNA hybridizable with a preproenkephalin B cDNA probe has been found in the porcine spinal cord and ileum besides the hypothalamus, and these RNA species exhibit an indistinguishable size. The results presented indicate that each opioid peptide precursor is synthesized in different tissues.
Effects of (2R, 5R)‐6‐heptyne‐2,5‐diamine, a potent inhibitor of l‐ornithine decarboxylase, on rat hepatoma cells cultured in vitro
Authors: MAMONT Pierre S. , SIAT Marlyse , JODER‐OHLENBUSCH Anne‐Marie , BERNHARDT Annie , CASARA Patrick
Published: March 2005

dl‐α‐Difluoromethylornithine (F2MeOrn), the most widely‐used inhibitor of l‐ornithine decarboxylase, has been a useful tool to demonstrate that polyamine biosynthesis is required to maintain maximum rates of cell proliferation. However, in most eukaryotic cell systems, F2MeOrn exerts cytostatic rather than cytotoxic effects. This may be due to the fact that this inhibitor creates only incomplete polyamine deficiency. In particular, F2MeOrn scarcely depletes intracellular spermine levels.

We now demonstrate in rat hepatoma tissue culture (HTC) cells that (2R, 5R)‐6‐heptyne‐2,5‐diamine, a more potent irreversible inhibitor of l‐ornithine decarboxylase than F2MeOrn, decreases the concentrations of all polyamines including spermine. In parallel with the depletion of these amines, there is a progressive decrease in the rate of cell proliferation and in cell viability. Restoration of the intracellular polyamine content, by addition to the medium of polyamines or a high concentration of l‐ornithine, the substrate of l‐ornithine decarboxylase, further demonstrates that the antiproliferative effects of (2R, 5R)‐6‐heptyne‐2,5‐diamine do result from polyamine deficiency.

These findings support the concept that polyamines play an essential function in the cell division processes and emphasize the vital function of spermine in mammalian cells.
Location of the sulfhydryl groups involved in disulfide interaction between the neighboring proteins L6 and L29 in mammalian ribosomes
Authors: NIKA Heinz , HULTIN Tore
Published: March 2005

Proteins L6 and L29 occupy closely adjacent sites in mammalian 60‐S ribosomal subparticles and are easily cross‐linked by intermolecular disulfide bond formation. For locating the interacting thiols within the polypeptide chains the dissociated proteins L6 and L29 obtained from the isolated disulfide complex were subjected to S‐cleavage following [14C]cyanylation of the two cysteine residues. Four split products of the [14C]cyanylated proteins were isolated by dodecylsulfate gel electrophoresis. Two of these could be identified by autoradiography as the selectively labeled C‐terminal fragments. For unequivocal assignment of the fragments to the parent proteins, a simple and generally applicable method of cleaving cyanylated proteins in polyacrylamide gel for subsequent diagonal analysis was developed. The experiments indicated that the sulfhydryl group of L6 interacting with L29 is located at a distance of approximately 80 amino acid residues from the N‐terminus. In the intact ribosome this sequence contains a clostripain‐sensitive and trypsin‐sensitive portion of the protein more or less exposed at the ribosomal surface. In the case of protein L29, the interacting sulfhydryl group was located at a distance of approximately 40 amino acid residues from the C‐terminal.
Purification and characterization of oocyte vitellin from Perinereis cultrifera (polychaete annelid)
Authors: BAERT Jean‐Luc , SAUTIERE Pierre , PORCHET Maurice
Published: March 2005

Vitellin was identified and purified from submature oocytes of Perinereis cultrifera by concanavalin‐A–Sepharose and Ultrogel AcA 34 column chromatography. The yolk protein was defined as a glycolipoprotein with a relative molecular mass of 380000. Upon dissociation by sodium dodecyl sulphate, vitellin was shown to release five polypeptide subunits which ranged in relative molecular mass from 98000–16000. The purified vitellin was further characterized by amino acid analysis and its lipid and carbohydrate contents. The molecule contained about 5% carbohydrate and 16% lipid.

Antiserum prepared against vitellin was shown to react with a component from the coelomic fluid of maturing females. Thus, it was suggested that in nereid annelids, considered phylogenetically and morphologically primitive, oocyte differentiation might depend upon the incorporation of a vitellogenin as in insects and many oviparous vertebrates.
Tyrosine modification of glucose dehydrogenase from Bacillus megaterium
Authors: FRÖSCHLE Marion , ULMER Wolfgang , JANY Klaus‐Dieter
Published: March 2005

The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residue/subunit. The nitration does not influence the dissociation‐reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP.

The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr‐254 in the covalent structure of the enzyme.

After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer.

Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr‐254 which is essential for the catalytic activity and the other one is Tyr‐160 which seems to be located in the subunit binding area.
Kinetic mechanism of a flavonol‐ring‐B O‐glucosyltransferase from Chrysosplenium americanum
Authors: KHOURI Henry , IBRAHIM Ragai K.
Published: March 2005

The kinetic mechanism of a flavonol‐ring‐B O‐glucosyltransferase from Chrysosplenium aniericanum was investigated. Substrate interaction kinetics for the flavonol and UDPG gave converging lines, which were consistent with a sequential bireactant binding mechanism. They also showed substrate inhibition with respect to the flavonol. Intercept and slope replots were linear and gave a KA of 250μM and a KB of 10μM. Product‐inhibition studies showed competitive inhibition between UDPG and UDP (KiQ 20 μM) and non‐competitive inhibition between the flavonol substrate and its glucoside (KiP 1 mM). Kinetic patterns were consistent with an ordered bi‐bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP is the final product released. The high KiP value, as compared with that of KB, indicates that the reaction is not inhibited by the glucosylated products formed and conforms with the accumulation of flavonol glucosides in C. americanum.
Regulation by adenosine of the vasopressin‐sensitive adenylate cyclase in pig‐kidney cells (LLC‐PK1L) grown in defined media
Authors: ROY Christian
Published: March 2005

LLC‐PK1L cells, a kidney‐derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10% fetal bovine serum, LLC‐PK1L cells had about 100‐times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10‐fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6‐L‐2‐phenylisopropyl‐adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladenosine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentration showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high‐affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added.

The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisopropyladenosine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high‐affinity component of the hormonal response could be demonstrated.
Purification and properties of aerobic and anoxic forms of pyruvate kinase from red muscle tissue of the channelled whelk, Busycotypus canaliculatum
Authors: PLAXTON William C. , STOREY Kenneth B.
Published: March 2005

Aerobic and anoxic variants of radular retractor muscle pyruvate kinase (PK‐aerobic and Pk‐anoxic) from the gastropod mollusc, Busycotypus canaliculatum, were purified to homogeneity and respective specific activities of 368 and 186 μmol of product min−1 mg protein−1. Both PK variants were apparent homotetramers with native molecular masses of about 235 kDa, but differed in several other physical characteristics including pI (5.81 ± 0.06 for PK‐aerobic, 5.42 ± 0.03 for PK‐anoxic) and chromatographic behavior on several columns used during their respective purifications. The two enzymes differed greatly in several kinetic properties. Affinity for phosphoenolpyruvate was more than tenfold greater for PK‐aerobic (K0.5= 0.067 ± 0.002 mM; h= 0.99 ± 0.10), whereas the cooperative effect for phosphoenolpyruvate binding was greatly enhanced for PK‐anoxic (K0.5= 0.85 ± 0.02 mM, h= 2.57 ± 0.01). Although the affinities for the second substrate, ADP, were identical for both enzyme forms (apparent Km= 0.25 mM) pK‐anoxic showed greater substrate inhibition by high concentrations of ADP. Likewise, affinities for K+ and Mg2+ were similar but PK‐anoxic showed a greater degree of cooperativity with Mg2+ (h= 2.50 ± 0.02) than did PK‐aerobic (h= 1.70 ± 0.06). Saturating concentrations of fructose 1,6‐bisphosphate (50 μM) activated PK‐anoxic resulting in an enzyme with properties similar to fructose‐1,6‐bisphosphate‐activated PK‐aerobic, with K0.5 values for phosphoenolpyruvate of about 0.04 mM and Hill coefficients of 1.1. PK‐anoxic showed much stronger regulation by the allosteric inhibitors MgATP, phenylalanine, proline and alanine. Fructose 1,6‐bisphosphate partially relieved the inhibitions by ADP, MgATP, alanine, proline and arginine phosphate of both enzyme forms. However, at 0.1 mM phosphoenolpyruvate PK‐aerobic was much more sensitive to activation by fructose 1,6‐bisphosphate, Ka values being 0.05 ± 0.01 μM for PK‐aerobic and 1.3 ± 0.1 μM for PK‐anoxic. In the presence of 1.0 mM alanine and 1.5 mM MgATP much higher concentrations of fructose 1,6‐bisphosphate were required for activation of PK‐anoxic (Ka= 5.2 ± 0.4μM) than for PK‐aerobic (Ka= 0.02 ± 0.01 μM). Variations in pH over the range likely occurring in vivo during anaerobiosis caused no significant additional kinetic differences between the two enzyme forms. The dissimilarity in kinetic properties of PK‐aerobic and PK‐anoxic indicate that red muscle PK activity is probably strongly depressed in vivo during anoxia stress.
Nucleotide sequence of cucumber‐mosaic‐virus RNA 2 reveals a translation product significantly homologous to corresponding proteins of other viruses
Authors: REZAIAN M. Ali , WILLIAMS Rhys H. V. , GORDON Karl H. J. , GOULD Allan R. , SYMONS Robert H.
Published: March 2005

The nucleotide sequence of the 3035 residues of RNA 2 (Mr 1.03 × 106) of the Q strain of cucumber mosaic virus (CMV) was determined by sequencing M13 clones of the RNA 2cDNA and by dideoxy sequencing using primers prepared either from M13 clones or by chemical synthesis. A single long open reading frame starts at the second AUG from the 5′ end of RNA 2 and encodes 839 amino acids (Mr 94333). This frame has flanking regions of 92 nucleotides at the 5′ terminus and 423 nudeotides at the 3′ terminus.

Computer analysis of the nucleotide sequence showed that CMV RNA 2 has a significant homology with RNA 2 of brome mosaic virus (BMV) and alfalfa mosaic virus (AMV) and also with a region for tobacco mosaic virus (TMV) RNA encoding the read‐through part of the 183‐kDa protein. About 400 amino acids in the central region of the CMV RNA 2 translation product have a striking homology with the corresponding proteins encoded by BMV and AMV and with the read‐through part of the TMV 183‐kDa protein. Hydrophobicity plots of CMV and BMV RNA translation products also had apparent similarities.

It is concluded that CMV is related to BMV, AMV and TMV in order of increasing evolutionary divergence.
Sequence‐dependent structural variation in single‐helical DNA Proton NMR studies of d(T‐A‐T‐A) and d(A‐T‐A‐T) in aqueous solution
Authors: MELLEMA Jan‐Remt , PIETERS Jane M. L. , VAN DER MAREL Gijs A. , VAN BOOM Jacques H. , HAASNOOT Cornelis A. G. , ALTONA Cornelis
Published: March 2005

The two deoxyribotetranucleoside triphosphates d(T‐A‐T‐A) and d(A‐T‐A‐T) were investigated in aqueous solution by one‐ and two‐dimensional proton NMR at 300 and 500 MHz. It is demonstrated that both compounds occur predominantly in the single‐helical form. Accurate coupling constants are obtained by computer simulation of several 500‐MHz spectra. The data are interpreted in terms of N and S pseudorotational ranges. The geometry of the major S‐type conformers displays a clear sequence dependence, as expressed by variation of the endocyclic backbone angle δ (C5′‐C4′‐C3′‐O3′). A simple sum rule is proposed to predict δ variation in single‐helical DNA fragments. Comparisons are made with other sequence‐dependent geometries as observed in a double‐helical B‐DNA fragment in the crystalline state.

Furthermore, one‐ and two‐dimensional nuclear Overhauser effect (NOE) spectroscopy was carried out on d(T‐A‐T‐A). An inventory is made of the observed intra‐ and inter‐residue NOEs. The NOE data confirm the presence of a highly stacked single‐helical conformation of d(T‐A‐T‐A) in solution. No indications are found for the formation of a bulge‐out structure as observed for analogous alternating purine‐pyrimidine oligoribonucleotides.
Characterization and properties of a 5S‐RNA‐protein complex released from heated 60S ribosomal subunits
Authors: REBOUD Anne‐Marie , DUBOST Simone , REBOUD Jean‐Paul
Published: March 2005

When rat liver 60S ribosomal subunits were heated in phosphate buffer in the presence of MgCl2, 5S RNA was released in the form of a nucleoprotein complex (RNPH), which was isolated either by electrophoresis in polyacrylamide gel or centrifugation through a sucrose gradient. In addition to L5 several proteins of functional significance were identified in the complex: the acidic phosphoproteins P1‐P2 and, as weaker spots, L3‐L4, L6‐L7 and L22. Most of these proteins were also found, but only as traces, in the RNPEDTA used as a control. RNPH was able to associate with 40S subunits. Our results support the interpretation that RNPH is located at the subunits' interface, at or near the peptidyl‐transferase center.
The amino acid sequence of GTP:AMP phosphotransferase from beef‐heart mitochondria
Authors: WIELAND Barbara , TOMASSELI Alfredo G. , NODA Lafayette H. , FRANK Rainer , SCHULZ Georg E.
Published: March 2005

The amino acid sequence of GTP:AMP phosphotransferase (AK3) from beef‐heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP:AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30% of all aligned residues being identical, the homology between AK3 and AK1 is well established. As derived from the known three‐dimensional structure of AK1, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N‐termini indicates enzyme processing.
A 20 × 103‐base mosaic gene identified on the mitochondrial chromosome of Podospora anserina
Authors: JAMET‐VIERNY Còrinne , BEGEL Odile , BELCOUR Leon
Published: March 2005

By DNA sequencing and hybridization experiments we have localized the genes cob and col on the mitochondrial chromosome of Podospora anserina. The positions we have determined for these two genes are different from those previously attributed to them. The presence in the gene col of at least two introns, belonging respectively to class I and II, has been demonstrated. This gene, with a size of about 20 × 103 bases, appears to be the longest known mitochondrial mosaic gene.
Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus
Authors: ZETTLMEISSL Gerd , TESCHNER Wolfgang , RUDOLPH Rainer , JAENICKE Rainer , GÄADE Gerd
Published: March 2005

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico‐chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable.

Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 ± 1500 for both native and denatured enzyme.

The unfolding transition at varying guanidine · HCl concentrations is characterized by a two‐step profile: at 0.4–0.8 M, partial unfolding is parallelled by inactivation; at 2.0–2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4–1.8 M guanidine · HCl, partial unfolding is superimposed by aggregation.

The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine · HCl. Changes in the far‐ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%.

Denaturation in 6 M guanidine · HCl, as monitored by the decrease of protein fluorescence, is fast (<8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (<20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long‐term denaturation, is determined by a single first‐order reaction characterized by τ= 29 ± 3 min (20°C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate‐limiting for reactivation after long‐term denaturation, are characterized by a high energy of activation (112 ± 8 kJ/mol). The slow reactivation step is compatible in rate with the first‐order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62–90].
Distinction of the roles of the two high‐affinity calcium sites in the functional activities of the Ca2+‐ATPase of sarcoplasmic reticulum
Authors: SCOTT Terrence L. , SHAMOO Adil E.
Published: March 2005

The effects of trypsin digestion and low temperature on Ca2+ binding and on Ca2+ activation of ATP hydrolysis by the high‐affinity transport sites of the Ca2+,Mg2+‐ATPase of sarcoplasmic reticulum were examined. Sarcoplasmic reticulum vesicles contain 0.7–1.1 high‐affinity Ca2+ sites per 105 g sarcoplasmic reticulum with K= 3–5 × 105 M−1, as well as sites of lower affinity. The first cleavage of the ATPase with trypsin (TD1) has no effect on the binding properties of the high affinity sites. The second tryptic cleavage (TD2) decreases the affinity of the high sites to K= 3 × 104 M−1 with conservation of the total number of sites.

The purified ATPase contains 1.6 – 2.0 high affinity Ca2+ sites per 105 g protein when measured at 23°C, while at 0–4°C there is ∼ 1 high‐affinity (K= 5 – 10 × 105 M−1) affinity site and ∼ 1 intermediate‐affinity (K= 3 × 104 M−1) site per 105 g. Trypsin digestion to the point of TD1 has no effect on either the number or the binding constants of the high‐affinity sites. Upon TD2 cleavage, one of the sites is converted to the intermediate‐affinity state, while the other remains at high affinity. After TD2 modification of the enzyme both of the sites are in the intermediate affinity state at 4°C.

On the basis of the binding data, several models for the roles of the Ca2+ sites in the activation of ATP hydrolysis are derived. The results are summarized by a scheme in which the two high‐affinity Ca2+ sites are heterogeneous with respect to sensitivity to temperature and to TD2 modification. The results of this and a previous study (Scott, T. L. and Shamoo, A. E. (1982) J. Membr. Biol. 64, 137–144] indicate that while occupation of either of the two Ca2+ sites can stimulate ATP hydrolysis, the site which is sensitive to TD2 is essential for the coupling of hydrolysis to Ca2+ transport.
Enzymatic glucosylation of dolichol monophosphate and transfer of glucose from isolated dolichyl‐D‐glucosyl phosphate to ceramides by BHK‐21 cell microsemes
Authors: SUZUKI Yasuo , ECKER Christian P. , BLOUGH Herbert A.
Published: March 2005

Enzymatic glucosylation of dolichol monophosphate (dolichol‐P) from UDP‐D‐[3H]glucose was studied using the microsomal fraction of BHK‐21 cells. The reaction product was separated by preparative thin‐layer chromatography, further purified by DEAE‐cellulose acetate column chromatography, and characterized as dolichyl‐β‐D‐glucosyl phosphate (Dol‐P‐Glc). The microsomal fraction of BHK cells catalyzed the incorporation of glucose from UDP‐[3H]glucose into ceramides (endogenous and exogenous) and Dol‐P; both reactions required Mn2+. Maximal glucosylation of Dol‐P was achieved at pH 5.6–5.8 in the presence of a non‐ionic detergent, Zonyl A. Glucosylation of exogenous Dol‐P, from UDP‐Glc, was non‐competitively inhibited by exogenous ceramides.

Incubation of Dol‐P‐[3H]Glc or Dol‐P‐[14C]Glc with liposomes (containing ceramides) and the microsomal fraction of BHK‐21 cells resulted in the formation of a radioactive glucolipid which comigrated with the same RF value as glucosylceramide (Glc‐Cer) on silica gel thin‐layer chromatography. Transfer of [14C]glucose from Dol‐P‐[14C]Glc to exogenous ceramides was confirmed by double‐labeling techniques. The pH dependence for transfer of radio‐labeled glucose from Dol‐P‐[3H]Glc to ceramides was multi‐phasic (optima at pH 4.0 and 7.0); glycosylation occurred within 5 min and Zonyl A was absolutely essential for the transfer reaction. These results indicate that Dol‐P‐Glc may also participate in the synthesis of ceramide hexosides.
Mechanism of hepatic megamitochondria formation by ammonia derivatives
Authors: WAKABAYASHI Takashi , HORIUCHI Michio , SAKAGUCHI Mikako , MISAWA Kazuko , ONDA Hiroyuki , HJIMA Moritake , ALLMANN David W.
Published: March 2005

Correlation between the chemical structure and the ability to induce hepatic megamitochondria formation was studied by feeding mice and rats diets containing a wide spectrum of ammonia derivatives.

Ammonia derivatives with electron‐releasing groups, such as hydrazine, phenylhydrazine, hydroxylamine and aniline were effective in inducing megamitochondria.

Ammonia derivatives with electron‐withdrawing groups, such as formamide, sulfamic acid, acetamide were ineffective in inducing megamitochondria.

Inducibility of ammonia derivatives with electron‐releasing groups plus electron‐withdrawing groups for the megamitochondria formation was dependent upon nucleophilicity of the chemical: 2,4‐dinitrophenylhydrazine induced megamitochondria, while acetanilide did not induce megamitochondria.

The megamitochondria formation induced by ammonia derivatives was a reversible process.

Freeze‐fracture studies on megamitochondria indicated that megamitochondria were formed by the fusion of adjacent mitochondria.

Phosphorylating capacity of megamitochondria (hydrazine‐induced megamitochondria, for example) were normal despite morphological changes.

These data might suggest that the nucleophilicity of chemicals plays a key role in the induction of hepatic megamitochondria. These data might also suggest that the phenomenon is an adaptive process to changes of intracellular milieu.
Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported post‐translationally in Escherichia coli
Authors: PAGES Jean‐Marie , ANBA Jamila , BERNADAC Alain , SHINAGAWA Hideo , NAKATA Atsuo , LAZDUNSKI Claude
Published: March 2005

Hyperproduction of phosphate‐binding protein, PhoS, in strains carrying a multicopy plasmic containing the phoS gene, resulted in saturation of export sites. As a consequence, pre‐PhoS was accumulated both in the inner membrane and in the cytoplasm. This was evidenced both in electron‐microscopy and after cell fractionation. Only the membrane‐associated precursor could be matured and exported. The signal sequence of the cytoplasmic pre‐PhoS was slowly degraded. It was first cleaved about in its middle and then completely removed. Electron microscope studies demonstrated that the cytoplasmic pre‐PhoS cannot be exported post‐translationally.
Methylamine‐induced conformational change of α2‐macroglobulin and its zinc(II) binding capacity
Authors: ÖSTERBERG Ragnar , MALMENSTEN Bo
Published: March 2005

Methylamine induces a conformational change of α2‐macroglobulin which is very similar to that obtained by proteinase reaction and binding. This was shown by small‐angle X‐ray scattering at 21 °C in 0.03 M Hepes buffer of pH 8.0 containing 0.15 M NaCl and 0.3 mM EDTA. When α2‐macroglobulin reacts with methylamine the side maximum virtually disappears from the X‐ray scattering curve and the radius of gyration decreases from 7.8 nm to 7.2 nm.

The X‐ray data of α2‐macroglobulin are consistent with an open shape model similar to that deduced via electron micrographs [Schramm, H. J. and Schramm, W. (1982) Hoppe‐Seyler's Z. Physiol. Chem. 363, 803–812]; one projection of the model resembles the letter H; the four subunits are mainly represented as elliptical cylinders which are connected via a central, quite flat cylinder.

Zinc(II) ions cause aggregation of α2‐macroglobulin even at such a low total zinc concentration as 12.5 μM; for 25 μM zinc(II) concentration, the average molecular mass indicates that the aggregation goes beyond the dimeric stage. Monomeric species of α2‐macroglobulin appear to have the capacity specifically to bind 8.0 zinc(II) ions per molecule, which corresponds to two zinc(II) ions per subunit.
A kinetic study of the oxidative deamination of L‐glutamate by Peptostreptococcus asaccharolyticus glutamate dehydrogenase using a variety of coenzymes
Authors: HORNBY David P. , ENGEL Paul C.
Published: March 2005

1. The NAD+ ‐specific glutamate dehydrogenase from Peptostreptococcus asaccharolyticus follows Michaelis‐Menten kinetics in contrast to the enzyme from several other sources, and thus gives linear double‐reciprocal plots of initial‐rate data.

2. The initial‐rate parameters have been determined for this bacterial glutamate dehydrogenase in the direction of oxidative deamination. The use of alternative coenzymes leads to some conclusions about the order of substrate addition.

3. An investigation of the pH dependence of this reaction reveals that the binding of oxidised coenzyme is independent of pH over the range 6–9.

4. The kinetic data are consistent with an ordered addition of coenzyme prior to glutamate, the reverse of the mechanism derived with ox glutamate dehydrogenase in the presence of ADP.
Biosynthesis of N‐acetylmannosaminuronic‐acid‐containing cell‐wall polysaccharide of Bacillus subtilis
Authors: ARAKAWA Hirohumi , ITO Eiji
Published: March 2005

The particulate enzyme from Bacillus subtilis AHU 1031 catalyzed the synthesis of a polysaccharide and glycolipids from UDP‐N‐acetylmannosaminuronic acid (UDP‐ManNAcUA), UDP‐N‐acetylglucosamine (UDP‐GlcNAc), and UDP‐glucose (UDP‐Glc). The polysaccharide synthesis required UDP‐ManNAcUA and UDP‐GlcNAc, proceeded optimally at pH 8.5 and in the presence of 5 mM MgCl2 and 2.5 mM dithiothreitol, and was stimulated by the addition of UDP‐Glc. The molar ratio of ManNAcUA, GlcNAc, and Glc incorporated into polysaccharide was calculated to be 1:1:1.8 from chemical analysis involving reduction with water soluble carbodiimide; its relative molecular mass was estimated to be 12000. The analysis of Smith degradation products revealed that the polysaccharide backbone is composed of repeating trisaccharide units comprising ManNAcUA, GlcNAc, and Glc. Based on the data regarding the time course of the incorporation of glucose into the polysaccharide, extra glucose seems to be attached to the polysaccharide backbone as lateral branches. The saccharide moieties of the glycolipids were identified as GlcNAc, ManNAcUA‐GlcNAc, and Glc‐ManNAcUA‐GlcNAc from several analytical criteria. The addition of antibiotic 24010, a tunicamycin‐like antibiotic, at 10μg/ml resulted in almost complete inhibition of the synthesis of glycolipids and polysaccharide. It is therefore concluded that the glycolipids function as intermediates in polysaccharide formation. Incubation of the ManNAcUA‐GlcNAc‐linked lipid. (labeled in the ManNAcUA moiety) with the particulate enzyme and UDP‐Glc resulted incorporation of radioactivity into a trisaccharide‐linked lipid and a polysaccharide. These results suggest that the particulate enzyme utilizes the trisaccharide moiety of the Glc‐ManNAcUA‐GlcN Ac‐linked lipid for the elongation of the main polysaccharide chain presumed to be cell wall acidic polysaccharide of this strain.
Actinomycin D, 1H NMR studies on intramolecular interactions and on the planarity of the chromophore
Authors: JURETSCHKE Hans Paul , LAPIDOT Aviva
Published: March 2005

The conformation of actinomycin D in acetone and chloroform solution at different temperatures has been studied by 1H NMR spectroscopy.

At lower temperature the resonances due to the two chromophoric amino protons were observed. These signals exhibit very different resonance positions indicating a severely hindered rotation of the 2‐amino group and the presence of a hydrogen‐bond connecting the 2‐amino and the 1‐carbonyl groups.

In 1H NMR spectra of partially 15N‐enriched actinomycin D, the 1JN‐H coupling constants at the 2‐amino group were determined and a strong sp2 character for the 2‐amino nitrogen was deduced. The strong amide character of the 2‐amino group is caused by mesomerism involving the 1‐carbonyl group.

The amino proton signals are sensitive indicators for differences in the spatial relationship of the diverse parts of the actinomycin molecule.

At lower temperatures a simultaneous and selective broadening of the α ring threonine and valine amide proton signals as well as of the 2‐amino group resonance was observed, indicating the presence of one dynamic process in the molecule which slows down upon temperature reduction. A swinging motion of the N(10) nitrogen through the chromophore plane would explain this observation.

The interpretation of these results requires the presence of a non‐planar chromophoric system in the actinomycin molecule in acetone and chloroform solution. The possible implications of this non‐planarity for the intercalation process and for the biological activity of the drug are discussed.
113Cd‐1H spin‐spin couplings in homonuclear 1H correlated spectroscopy of metallothionein
Authors: NEUHAUS David , WAGNER Gerhard , VAŠÁK Milan , KÄGI Jeremias H. R. , WÜTHRICH Kurt
Published: March 2005

Heteronuclear spin‐spin couplings between 113Cd and Cβ protons of the metal‐bound cysteines were observed in phase‐sensitive, double‐quantum filtered, homonuclear two‐dimensional correlated (COSY) 1H NMR spectra of 113Cd‐metallolhionein‐2 from rabbit liver. Comparison of 113Cd‐and 112Cd‐metallotriionein‐2 spectra revealed that 19 1H spin systems show heteronuclear couplings to at least one 113Cd and were thus identified as 19 of the 20 cysteines in this protein. From a detailed analysis of the manifestations of heteroouelear couplings in the homonuclear 1H COSY speclra, two cyslcincs could be identified as ‘bridging cysleines’, with spin‐spin couplings to two different 113Cd nuclei. The observed 113Cd‐1H coupling constants vary between ≤5 Hz and 80 Hz.