bluuebunny/crossref_metadata_2025_split huggingface dataset:
Yeast mitochondria contain a linear RNA strand complementary to the circular intronic bI1 RNA of cytochrome b
Authors: HALBREICH Avraham , GRANDCHAMP Claude , FOUCHER Marcelle 
Published: March 2005

bIl RNA (excised from the first intron of the long form of the cytochrome b gene of Saccharomyces cerevisiae mitochondria) hybridizes with the two strands of a Bg/II‐MboI DNA segment from this region. This fraction is resistant to digestions by DNase I and RNase T1 and disappears completely upon alkali hydrolysis. Strand‐specific labeling of an intronic DNA fragment, cloned in pBR322 plasmid, was accomplished through the use of a T4 DNA polymerase. The purity of the probes was demonstrated by cloning an exon‐intron fragment and labeling it by the, same procedure; mRNA and pre‐mRNA bands hybridized only with the transcribed DNA strand whereas bI1 RNA hybridized with the two strands under the stringent washing conditions employed (tm+20 °C).Several experimental results argue against the possibility that the observation of two complementary bI1 RNA strands results from a partial self‐complementarity of the RNA.a) A pre‐mRNA intermediate from a box8 (G5046) mutant, still containing this intron, hybridizes only with the transcribed DNA strand of the pure intronic probe.b) The amount of the non‐sense bI1 RNA strand is very low, in cells from two wild‐type strains, relative to the sense RNA strand during the early stages of growth on glucose. It increases as the cells are released from glucose repression.c) bI1 RNA is resistant to RNase.d) Very little self‐complementarity is seen by computer analysis of the sequence.Purified bI1 RNA is seen by electron microscopy under non‐denaturing conditions as a mixture of double‐stranded circular and linear molecules thus confirming the existence of the two complementary strands. The disappearance of all material following alkali hydrolysis demonstrates that these are indeed two RNA strands. Under fully denaturing conditions a mixture of single‐stranded circular and linear molecules is seen as reported previously (Cell, 19, 321–329, 1980). We conclude that yeast mitochondria contain the two complementary bI1 RNA strands, one circular and the other linear. Considering a largely asymmetrical transcription of the mitochondrial genome in yeast and assuming that circularization of some intronic RNAs is part of RNA processing, we do not believe that the two strands are each a mixture of linear and circular molecules.The ratio of non‐sense to sense bI1 RNA in a cytoplasmic petite mutant, A1B1, also varies according to growth conditions. It changes from 2.4 in galactose‐grown cells to 0.95 in glucose‐grown cells thus demonstrating that the effect of glucose repression on mtRNA metabolism may take place without a contribution from the mitochondrial genome.
Conformational adaptation of RTEM β‐lactamase to cefoxitin
Authors: CITRI Nathan , KALKSTEIN Abraham , SAMUNI Amram , ZYK Naomi 
Published: March 2005

Cefoxitin, a poor substrate of the RTEM β‐lactamase (penicillin amido‐β‐lactam hydrolase, EC 3.5.2.6), induces a reversible change in the conformation of the enzyme. The change is manifested in gradual loss of catalytic activity and increased susceptibility to proteolytic inactivation. It is prevented by antibodies, which stabilize the native conformation. By contrast, divalent cations, which have no effect on the native enzyme, delay recovery from the cefoxitin‐induced state, presumably by reacting with sites made accessible in the partly unfolded enzyme. Prolonged exposure to excess of cefoxitin causes a similar delay.The kinetic evidence, namely, the initial burst of consumption of cefoxitin and the subsequent gradual recovery of activity with better substrates, appears to be consistent with acylation of the active site by cefoxitin followed by a slower deacylation step [Fisher et al. (1980) Biochemistry 19, 2895–2901]. However, additional evidence leads us to conclude that the kinetics observed reflect deformation of the active site, rather than its blockage, by cefoxitin. Of most significance is the transient change in specificity, i. e. a preferential interaction of the recovering enzyme with substrates which are closest in structure to cefoxitin.
Photoaffinity labelling of dopamine receptors
Authors: WOUTERS Walter , VAN DUN Jacky , LADURON Pierre M. 
Published: March 2005

A new compound, azapride, i.e. 4‐azido‐5‐chloro‐2‐methoxy‐ N‐[1‐(phenylmethyl)‐4‐piperidinyl]benzamide, the azide derivative of the dopamine antagonist clebopride, was synthesized and tested for its usefulness as a photoaffinity probe for dopamine receptors. Without photolysis the azide behaved as a very selective dopamine antagonist. When a microsomal preparation of dog striatum was pretreated with the azide and subsequently irradiated with ultraviolet light, dopamine receptors became irreversibly blocked. This reaction was dependent on both azide concentration and time of irradiation. The irradiation‐induced inhibition was quite selective for the dopamine receptor; various other receptor systems were not affected. Moreover the irreversible inhibition of dopamine receptors from dog striatum could be prevented by dopamine antagonists as well as by a dopamine agonist, but not by a serotonin antagonist. The irradiation‐induced inactivation by azapride remained unchanged in the presence of the scavenger p‐aminobenzoic acid.These findings provide evidence that azapride labels dopamine receptors specifically and irreversibly and by a true photoaffinity mechanism. It should therefore be of great value for further molecular characterization and purification of dopamine receptors.
l‐Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium
Authors: TAGUCHI Hayao , MATSUZAWA Hiroshi , OHTA Takahisa 
Published: March 2005

Heat‐stable and fructose‐1,6‐bisphosphate‐activated l‐lactate dehydrogenase (EC 1.1.1.27) has been purified from an extremely thermophilic bacterium, Thermus caldophilus GK24 [Taguchi, H., Yamashita, M., Matsuzawa, H. and Ohta, T. (1982) J. Biochem. (Tokyo) 91, 1343–1348]. N‐terminal sequence analysis of the first 34 amino acids of the enzyme indicates that the N‐terminal arm region (first 1–20 residues) known for the vertebrate l‐lactate dehydrogenases is completely missing in the T. caldophilus enzyme, while there is a high homology of sequence between the regions which are considered to be part of the NAD‐binding domain. The C‐terminal amino acid of the enzyme was phenylalanine. Analysis of the amino acid composition showed that T. caldophilus enzyme contained much more arginine and fewer lysine than other bacterial and vertebrate l‐lactate dehydrogenases.On modification reaction with 2,3‐butanedione in the presence of NADH and oxamate, an enhanced activity of the T. caldophilusl‐lactate dehydrogenase was obtained independently of fructose 1,6‐bisphosphate, and the modified enzyme was desensitized to fructose 1,6‐bisphosphate. Amino acid analysis indicated that such a desensitization in the active state was caused by the modification of only one arginine residue per the enzyme subunit. Desensitization of the enzyme was inhibited in the presence of fructose 1,6‐bisphosphate. A similar desensitization was observed using 1,2‐cyclohexanedione instead of 2,3‐butanedione. The enzyme was irreversibly modified with 2,3‐butanedione and characterized. The irreversibly modified enzyme also showed an enhanced activity independently of fructose 1,6‐bisphosphate, and its pyruvate saturation curve was similar to that of the native enzyme measured in the presence of fructose 1,6‐bisphosphate. Fructose 1,6‐bisphosphate, which increases the thermostability of the native enzyme, did not affect that of the modified enzyme, while thermostability of the modified enzyme slightly decreased. Amino acid analysis indicated that only the arginine content was decreased by the modification. These results show that arginine residue(s) exist in the binding site for fructose 1,6‐bisphosphate on the enzyme, and that the arginine residue(s) play some important role in the allosteric regulation of the enzyme activity.
The nature of the interaction of eukaryotic initiation factor 2 with double‐stranded RNA
Authors: ITAMAR Daliah , GONSKY Rivkah , LEBENDIKER Mario , KAEMPFER Raymond 
Published: March 2005

In addition to binding messenger RNA molecules at specific sequences, eukaryotic initiation factor 2 (eIF‐2) also binds to double‐stranded RNA (dsRNA). The dsRNA is a powerful inhibitor of initiation of eukaryotik translation, causing the inactivation of eIF‐2, but in the presence of certain mRNA templates, dsRNA fails to establish inhibition. Such mRNA templates bind to eIF‐2 with higher affinty than does dsRNA, while globin mRNA, a template sensitive to inhibition, binds with lower affinity.Here, the nature of the interaction between dsRNA and eIF‐2 was studied by examining both the binding of eIF‐2 to Penicillium chrysogenum dsRNA molecules carrying 32P label at their 5′ ends, and the ability of eIF‐2 to protect such label against pancreatic ribonuclease digestion. The results reveal binding sites for eIF‐2 at the 5′ ends, as well as throughout internal regions of the dsRNA molecule. At least 15 molecules of eIF‐2 can be accommodated on a 3000‐base molecule of P. chrysogenum dsRNA eIF‐2 protects a 105‐base‐pair 5′‐terminal fragment in dsRNA against digestion, but exhibits no noticeable preference for the 5′ ends. By contrast, eIF‐2 fails to protect label at the 5′ ends of denatured dsRNA molecules, even though it binds to them at internal sites more avidly than to native dsRNA.Binding of eIF‐2 to dsRNA is not restricted to specific sequences: eIF‐2 binds with equal affinity to the synthetic dsRNA sequence, poly(rI·rC). The data support the interpretation that eIF‐2 recoginzes the A conformation in dsRNA rather than sequence. Apparently binding of eIF‐2 at sites spaced 200 base pairs apart prevents relaxation of the intervening length of the double helix, thereby stabilizing the dsRNA molecule against ribonuclease attack.These results show that, even though dsRNA and mRNA compete in their binding to eIF‐2, the structural features recognized by eIF‐2 in these RNA species are distinct.
RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor
Authors: MIKULÍK Karel , JANDA Ivan , WEISER Jaroslav , ŠŤASTNÁ Jana , JIRÁŇOVÁ Anna 
Published: March 2005

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time‐ordered events. RNA and protein synthesis started during the first 5 min and net DNA syntesis at 60–70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4–5‐S RNA were synthesized.Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C‐labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.
Kinetic studies on surface‐mediated activation of bovine factor XII and prekallikrein
Authors: SUGO Teruko , KATO Hisao , IWANAGA Sadaaki , TAKADA Katsumi , SAKAKIBARA Shunpei 
Published: March 2005

The kaolin‐mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high‐Mr kininogen and kalin surface on the kinetics of these activation reactions were studied. High‐Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high‐Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 − 107 s−1· M−1, respectively.The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high‐Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 μM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 − 104 to 1.1 − 106 s−1· M−1. These results suggest that high‐Mr kininogen accelerates the surface‐mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.
The effects of terbium (III) on the Ca‐activated, K channel found in the resealed human erythrocyte membrane
Authors: WOOD Phillip G. , MUELLER Hannelore 
Published: March 2005

Incorporated terbium, Tb3+, activates the Ca‐activated K channel found in the resealed erythrocyte ghost membrane and allows the net efflux of K. As in the case of Ca activation, low levels of external K stimulate the net efflux of K. The action of incorporated Tb3+ seems to be analogous to that of incorporated Ca.Externally applied Tb3+, however, inhibits the net efflux of K after either Ca or Tb3+ activation. The net‐efflux of K can be inhibited by Tb3+ even after partial digestion of the channel by low levels of incorporated trypsin. Furthermore, the channel when incorporated into planar bilayers, can be inhibited by Tb3+. Externally applied Tb3+ does not seem to inhibit net K efflux indirectly via inhibition of the pathways for the co‐transported anions. In addition because of the low concentrations of Tb3+ required for inhibition, it seems unlikely that an alteration of surface potential is responsible for the observed effects.
Formation of carbon monoxide from CO2 and H2 by Methanobacterium thermoautotrophicum
Authors: EIKMANNS Bernhard , FUCHS Georg , THAUER Rudolf K. 
Published: March 2005

Cell suspensions of Methanobacterium thermoautotrophicum were found to reduce CO2 with H2 to CO at a maximal rate of 100 nmol − min−1− mg protein−1. Half‐maximal rates were obtained at a H2 and a CO2 concentration in the gas phase of 10% and 30%, respectively. The CO concentration in the gas phase surpassed the equilibrium concentration by a factor of more than 15 which indicates that CO2 reduction with H2 to CO was energy‐driven. This was substantiated by the observation that the cells only formed CO when they also generated methane and that CO formation was completely inhibited by uncouplers.CO formation by cell suspensions and by growing cells was inhibited by cyanide. Neither methane formation nor the electrochemical proton potential were affected by this inhibitor. Cyanide was shown to inactivate specifically the carbon monoxide dehydrogenase present in M. thermoautotrophicum. It is therefore concluded that reduction of CO2 to CO is catalyzed by this enzyme.CO production by growing cells was 5–10‐times slower than by resting cells. This is explained by effective CO assimilation in growing cells; when CO assimilation was inhibited by propyl iodide the rate of CO production immediately increased more than tenfold.
Affinity labeling of rabbit muscle fructose‐1,6‐bisphosphate aldolase with 5′‐[p‐(fluorosulfonyl)benzoyl]‐1, N6‐ethenoadenosine
Authors: PALCZEWSKI Krysztof , HARGRAVE Paul A. , FOLTA Ewa J. , KOCHMAN Marian 
Published: March 2005

Aldolase contains one tight binding site and one weak binding site per subunit for ATP [Kasprzak, A. and Kochman, M. (1980) Eur. J. Biochem. 104, 443–450]. The reaction of the ATP analog 5′‐[p‐(fluorosulfonyl)benzoyl]‐1, N6‐ethenoadenosine with rabbit aldolase A results in linear inactivation of enzyme with respect to covalent linkage of fluorescent label. The enzyme is completely protected against modification in the presence of saturating covalent binding (k2= 0.033 min−1) is preceded by a fast reversible binding step (Ki= 6.8 mM).Chemical modification of aldolase leads to formation of stable Nɛ(4‐carboxybenzenesulfonyl‐lysine (Cbs‐Lys) and O‐(4‐carboxybenzenesulfonyl‐tyrosine (Cbs‐Tyr) derivatives. Almost all Cbs‐Lys was found in the N‐terminal CNBr peptide (CN‐1), whereas Cbs‐Tyr was present both in the N‐terminal (CN‐1) and C‐terminal (CN‐2) peptide. From carboxypeptidase digestion and tryptic peptide analysis, Cbs‐Lys was localized in position 107, a small part of Cbs‐Tyr was detected in position 84, and the majority of Cbs‐Tyr was found in the C‐terminal position Tyr‐363.We conclude that the covalent binding of the ATP analog occurs at the mononucleotide tight‐binding site of aldolase and is associated with modification of Lys‐107 and Tyr‐363. This conclusion is based on the measurements of enzymatic activity loss as a function of ATP analog incorporation as well as on previous data. It is postulated that Lys‐107, which is the C‐6 phosphate binding site for fructose‐1,6‐P2, is in close proximity to the functionally important Tyr‐363. The rather small extent of modification of Tyr‐84 (0.15 mol/subunit), is due either to nonspecific protein modification or labeling of the weak mononucleotide binding site.
Direct assessment of β‐adrenergic receptors in intact rat adipocytes by binding of [3H]CGP 12177
Authors: LACASA Daniéle , AGLI Brigitte , GIUDICELLI Yves 
Published: March 2005

The characteristics of the binding of the hydrophilic β‐adrenergic antagonist [3H]CGP 12177 to intact rat adipocytes were studied at 37°C and 6°C. At both temperatures and at 90% saturation, the non‐specific binding was less than 30% of the total binding.At 37°C, specific [3H]CGP 12177 binding was rapid, reversible of high affinity (1.8 ± 0.4 nM) and saturable. The number of specific binding sites per adipocyte increased with the fat cell size (about 35000 and 115000 sites per cell in adipocytes with diameters of 60 μm and 88 μm, respectively) but remained constant when expressed per unit fat cell surface area. Displacement of [3H]CGP 12177 bound to adipocytes by unselective and selective β‐antagonists was stereospecific, had the same characteristics as those found in adipocyte membranes and showed a heterogeneous specificity for β1 and β2 adrenergic subtypes. In contrast, β agonist competition curves, Which modeled to two affinity‐states of binding, showed high‐affinity‐state Kd values for agonists 10–25‐times higher than those found in membranes under the same experimental conditions.At 6°C, although the number and affinity of the specific binding sites for [3H]CGP 12177 were the same as those found at 37°C, the Kd value for (–)‐isoproterenol binding to the high affinity state of these sites (3.0±0.5 nM) was 25‐times lower than at 37°C and similar to the value found in membrane preparations (1.5–4 nM).These results show that the [3H]CGP 12177 specific binding sites detected on intact adipocytes represent the physiological β‐adrenergic receptors. Moreover, this study extends to the adipocyte the validity of the model recently proposed for other cell lines, according to which in intact cells, but not in membranes, agonist‐binding promotes a rapid and temperature‐dependent conformational change of the β‐adrenergic receptors, leading to a progressive loss of capacity of agonists to form a high‐affinity complex.
Nucleotide sequence of yeast gene CPA1 encoding the small subunit of arginine‐pathway carbamoyl‐phosphate synthetase
Authors: WERNER Michel , FELLER André , PIÉRARD André 
Published: March 2005

A yeast DNA fragment carrying the gene CPA1 encoding the small subunit of the arginine pathway carbamoylphosphate synthetase has been sequenced. Only one continuous coding sequence on this fragment was long enough to account for the presumed molecular mass of CPA1 protein product. It codes for a polypeptide of 411 amino acids having a relative molecular mass, Mr, of 45358 and showing extensive homology with the product of carA, the homologou Escherichia coli gene. CPA1 and carA products are glutamine amidotransferases which bind glutamine and transfer its amide group to the large subunits where it is used for the synthesis of carbamoyl‐phosphate. A comparison of the amino acid sequences of CPA1 polypeptide with the glutamine amidotransferase domains of anthranilate and p‐aminobenzoate synthetases from various sources has revealed the presence in each of these sequences of three highly conserved regions of 8, 11 and 6 amino acids respectively. The 11‐residue oligopeptide contains a cysteine which is considered as the active‐site residue involved in the binding of glutamine. The distances (number of amino acid residues) which separate these homology regions are accurately conserved in these various enzymes. These observations provide support for the hypothesis that these synthetases have arisen by the combination of a common ancestral glutamine amidotransferase subunit with distinct ammonia‐dependent synthetases. Little homology was detected with the amide transfer domain of glutamine phosphoribosyldiphosphate amidotransferase which may be the result of a convergent evolutionary process.The flanking regions of gene CPA1 have been sequenced, 803 base pairs being determined on the 5′ side and 382 on the 3′ side. Several features of the 5′‐upstream region of CPA1 potentially related to the control of its expression have been noticed including the presence of two copies of the consensus sequence d(T‐G‐A‐C‐T‐C) previously identified in several genes subject to the general control of amino acid biosynthesis.