bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of action of an antiprogesterone, RU486, in the rabbit endometrium
Authors: RAUCH Michel , LOOSFELT Hugues , PHILIBERT Daniel , MILGROM Edwin
Published: March 2005

RU486 is a recently described antiprogesterone. In order to be able to understand its mechanism of action it is necessary to analyze its effect on a discrete gene product. We show here that the induction of uteroglobin mRNA by progesterone in the rabbit endometrium may be a suitable model for such studies since RU486 totally inhibits this effect without itself exerting any agonistic activity. Moreover, RU486, which does not bind to the estrogen receptor and is devoid of general antiestrogenic activity, partially inhibits the induction by estradiol of uteroglobin mRNA.

Studies of the interaction between [3H]RU486 and the progesterone receptor have been undertaken with the aim of understanding the antagonistic effect of this compound. The binding to DNA‐cellulose of heat‐activated [3H]RU486‐receptor complexes was slightly decreased (37%) when compared with that of the agonist [3H]R5020‐receptor complexes (47%). Detailed analysis of this difference showed that it was due to both a decreased activation of complexes and to a diminished affinity of activated complexes towards DNA. The change in activation was shown by the fact that at high concentrations of DNA, where all activated complexes are bound, agonist‐receptor complexes were bound to DNA in higher proportion than antagonist‐receptor complexes. Moreover a difference was also observed when studying the binding of agonist‐receptor and antagonist‐receptor complexes to charged resins (phosphocellulose, DEAE‐cellulose) which are known to discriminate between activated and non‐activated complexes.

Decreased affinity to DNA of antagonist‐receptor complexes was shown by studying their binding at various concentrations of DNA, either in crude cytosol or after isolating a homogenous population of activated‐receptor complexes by DNA‐cellulose chromatography and by comparing the salt extraction from DNA‐cellulose of agonist‐receptor and antagonist‐receptor complexes.

Both effects (decreased activation and diminished affinity towards DNA) were relatively moderate and could account only for a small decrease in the agonistic activity of RU486. Thus, the fact that this compound is a complete antagonist without any agonistic activity can only be explained by a defect in some further step of hormone action as, for instance in the specific interaction with the regulatory regions of the uteroglobin gene.

No immunological difference could be detected between [3H]R5020‐receptor and [3H]RU486‐receptor complexes, both interacted with the five monoclonal antibodies raised against purified R5020‐receptor complexes. This suggests that the difference in activity is not reflected by major structural differences between these two complexes.
Purification and molecular characterization of bovine pregastric lipase
Authors: BERNBÄCK Stefan , HERNELL Olle , BLÄCKBERG Lars
Published: March 2005

A pregastric lipase was purified from calf pharyngeal tissues. The purification procedure was based on chromatographies on octyl‐Sepharose and lentil‐lectin‐Sepharose followed by gel filtration. The final preparation, with an overall recovery of 26% of activity, gave a single protein band on dodecyl sulfate/polyacrylamide gel electrophoresis with a Mr of 55000. The Mr on gel filtration was 44–48000. The discrepancy may be due to the fact that pregastric lipase is a glycoprotein containing ∼ 10% (w/w) of carbohydrate. The pI was around 7.0 and the enzyme protein is characterized by a high content of branched, aliphatic amino acid residues. The NH2‐terminal amino acid sequence is: H2N‐Phe‐Leu/(Ile)‐Gly‐. Rabbit antibodies to the purified preparation detected only one component in the crude starting material in immuno‐blotting experiments. Preincubation with antiserum resulted in loss of enzyme activity, showing that the antibodies were directed against the lipase.
The occurrence, subcellular localization and partial purification of diamine acetyltransferase in the yeast Candida boidinii grown on spermidine or putrescine as sole nitrogen source
Authors: HAYWOOD Geoffrey W. , LARGE Peter J.
Published: March 2005

The yeast Candida boidinii when grown on spermidine, diaminopropane, putrescine or cadaverine as sole nitrogen source contains an N‐acetyltransferase capable of acetylating the primary amino groups of spermine, spermidine, acetylspermidines, acetylputrescine and α,ω‐diaminoalkanes. In the case of spermidine, the products were N1‐acetylspermidine and N8‐acetylspermidine in the ratio 50:45 with traces of other unidentified products. The enzyme was partially purified and the stoichiometry determined, together with apparent Km and V values for a number of substrates. The pH optimum was about 8.8 for putrescine and 9.3 for spermidine. The unstable enzyme was partially stabilized by 10% (v/v) glycerol or bovine serum albumin (5 mg/ml): The kinetic parameters were determined with putrescine as substrate and the mechanism shown to be of the sequential type. The enzyme was shown to be located in the mitochondria of C. boidinii, in contrast to mammalian N‐acetyltransferases. The enzyme was found in a number of other yeast species when grown on spermidine or putrescine, but was only present in those species that had previously been found to contain polyamine oxidase. It is suggested that in C. boidinii, as in mammals, acetylation of spermidine and putrescine must precede their catabolism.
Peroxisomal β‐oxidation system of Candida tropicalis
Authors: MORENO DE LA GARZA Myrthala , SCHULTZ‐BORCHARD Ursula , CRABB John W. , KUNAU Wolf‐H.
Published: March 2005

1. A multifunctional protein from oleate‐grown cells of Candida tropicalis has been purified and partially characterized. A simple two‐step purification has been developed involving ion‐exchange chromatography followed by dye‐ligand chromatography on blue Sepharose CL‐6B. Homogeneous enzyme with a subunit Mr of 102000 is obtained in 60% yield. The native relative molecular mass, determined by three different methods, yielded values which suggest that the enzyme is dimeric. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein revealed a single polypeptide band and reverse‐phase high‐performance liquid chromatography indicated a single component suggesting that this protein may consist either of two identical or very similar subunits.

2. Three β‐oxidation activities, enoyl‐CoA hydratase, 3‐hydroxyacyl‐CoA dehydrogenase and 3‐hydroxyacyl‐CoA epimerase, co‐purified with this protein. The ratio of the three β‐oxidation enzyme activities remained constant during purification and was unchanged by additional chromatographic methods (adsorption and affinity chromatography), thus indicating the multifunctional nature of this protein. Enzymatic staining of the purified protein for 3‐hydroxyacyl‐CoA dehydrogenase and epimerase, following electrophoresis in a polyacrylamide density gradient, further supported the multifunctionality of this protein.

3. After isopycnic centrifugation of a particulate fraction from oleate‐grown cells in a linear sucrose gradient the activities of all individual β‐oxidation enzymes cosedimented with catalase and with the glyoxylate bypass enzymes. This result demonstrated the peroxisomal localization of the multifunctional enzyme.

4. The relationship of this multifunctional protein to the two bifunctional β‐oxidation enzymes isolated from peroxisomes of rat liver and from glyoxysomes of cucumber seeds is discussed.
A new type of cereal lectin from leaves of couch grass (Agropyrum repens)
Authors: CAMMUE Bruno , STINISSEN Hetty M. , PEUMANS Willy J.
Published: March 2005

Extracts from couch grass (Agropyrum repens) leaves contain relatively high lectin concentrations. Preliminary experiments with crude extracts indicated that the leaf lectin differs from the embryo lectin of the same species and other Gramineae embryo lectins with respect to its sugar and blood group specificity, and serological properties. A comparison of the biochemical, physicochemical and biological properties of purified lectins from couch grass leaves and embryos, and wheat germ agglutinin revealed that the leaf lectin has the same molecular structure as the embryo lectins. It is a dimer composed of two identical subunits, which, however, are slightly larger than embryo lectin subunits. Structural differences between both couch grass lectins were further inferred from in vitro subunit exchange experiments and serological analyses. Whereas the embryo lectin readily forms heterodimers with embryo lectins from other cereal species and also is serologically indistinguishable from them, the leaf lectin does not exchange subunits with the same embryo lectins and is serologically different. In addition, couch grass leaf lectin exhibits specificity for N‐acetylgalactosamine and agglutinates preferentially blood‐group‐A erythrocytes whereas the embryo lectin is not inhibited by N‐acetylgalactosamine and exhibits no blood‐group specificity. It was observed also that the lectin content of couch grass leaves varies enormously during the seasons.
Rat liver glutaminase
Authors: McGIVAN John , VADHER Mita , LACEY Jean , BRADFORD Norah
Published: March 2005

Partially purified rat liver mitochondrial glutaminase shows a sigmoidal dependence on glutamine concentration, and an absolute requirement for inorganic phosphate as activator. Reconstitution with a mitochondrial membrane fraction changes the kinetic properties of the enzyme making the glutamine dependence more hyperbolic and reducing the concentration of phosphate required for half‐maximum activation. Glutaminase activity in isolated mitochondria is known to be increased as a result of mitochondrial swelling. In mitochondria suspended in isotonic medium, the properties of glutaminase resemble of the isolated enzyme while in swollen mitochondria the kinetic properties revert to those exhibited by the enzyme in association with the mitochondrial membrane.

It is postulated that mitochondrial glutaminase is regulated in situ by reversible association with the inner mitochondrial membrane which is mediated by mitochondrial swelling. This mechanism may explain the short‐term hormonally induced activation of the enzyme observed in isolated hepatocytes.
Flavobacterium heparinum 3‐O‐sulphatase for N‐substituted glucosamine 3‐O‐sulphate
Authors: BRUCE John S. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B.
Published: March 2005

A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3‐O‐sulphate from 2‐deoxy‐2‐sulphamido‐3‐O‐sulpho‐d‐glucose and 2‐acetamido‐2‐deoxy‐3‐O‐sulpho‐d‐glucose.

The activity was purified 10800‐fold by chromatography successively on CM‐Sepharose CL‐6B, hydroxy‐apatite, taurine‐Sepharose CL‐4B and CM‐Sepharose CL‐6B. Sodium dodecylsulphate/polyacrylamide gel elec‐trophoresis showed the enzyme to be homogeneous and of relative molecular mass 56000. Two novel assays were developed using 2‐[14C]acetamido‐2‐deoxy‐3‐O‐sulpho‐d‐glucose and 2‐deoxy‐2‐sulphamido‐3‐O‐sulpho‐d‐glucose as respective substrates.

The purified 3‐O‐sulphatase was shown to be free of all other known heparin‐degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N‐acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2‐fold enhancement of activity was effected by 0.002 mol dm−3 Ca2+. Inorganic phosphate and sulphate inhibited 3‐O‐sulphatase activity. The Km value of the N‐acetylated substrate was determined to be 42 μmol dm−3. No activity was detected with 2‐amino‐2‐deoxy‐3‐O‐sulpho‐d‐glucose.
Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P‐450scc incorporated into vesicles prepared from these phospholipids
Authors: TUCKEY Robert C. , STEVENSON Patricia M.
Published: March 2005

Cytochrome P‐450scc, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues, can be incorporated into artificial phospholipid vesicles and cholesterol binding to the cytochrome is affected by the composition of the vesicles. We have purified the phospholipids from the inner mitochondrial membrane fraction of the bovine corpus luteum where the cytochrome is located. The composition in mol % was 49% phosphatidylcholine, 34% phosphatidylethanolamine, 8.7% cardiolipin, 6.4% lysophosphatidylethanolamine and 1.5% phosphatidylinositol. The ratio of cholesterol to phospholipid (mol/mol) in the inner membrane fraction was 0.14 to 1. The Km for cholesterol of purified luteal cytochrome P‐450scc incorporated into vesicles prepared from the total inner mitochondrial membrane phospholipids was 0.063 mol of cholesterol per mol of phospholipid. Removal of the cardiolipin component of the inner mitochondrial membrane phospholipids prior to preparation of vesicles caused a four fold increase in the Kd of cytochrome P‐450 for cholesterol and a two fold increase in Km. The data suggests that in the inner mitochondrial membrane of the bovine corpus luteum the cholesterol concentration is less than saturating for cytochrome P‐450scc.
Purification of the 25‐kDa Vibrio cholerae major outer‐membrane protein and the molecular cloning of its gene: ompV
Authors: STEVENSON Gordon , LEAVESLEY David I. , LAGNADO Cathy A. , HEUZENROEDER Michael W. , MANNING Paul A.
Published: March 2005

The 25‐kDa peptidoglycan‐associated outer‐membrane protein and most likely porin of Vibrio cholerae is a major immunogenic species. It has been purified by ion‐exchange elution on hydroxyapatite followed by gel filtration on Bio‐Gel P150 both in the presence of sodium dodecyl sulfate. This protein, of greater than 90% purity as judged by Western blotting, has been used to raise antibodies in rabbits. The antisera were then used to screen V. cholerae gene banks, constructed in Escherichia coli K12, and this has enabled us to isolate several colonies harbouring the cloned gene. The plasmids in these colonies have been designated pPM451, pPM455 and pPM472. These plasmids have a 5.3 × 103‐base BamHI fragment of V. cholerae DNA in common. Restriction endonuclease mapping of these plasmids has been performed and the protein identified both by Western blot analysis and in E. coli K12 minicells. The protein is not efficiently expressed in E. coli K12. It is proposed to use the name ompV to describe the structural gene, present in the cloned DNA, for this V. cholerae outer membrane protein.
Uptake and processing of liposomal phospholipids by Kupffer cells in vitro
Authors: DIJKSTRA Jan , VAN GALEN Mieke , REGTS Djoeke , SCHERPHOF Gerrit
Published: March 2005

We investigated the intracellular metabolic fate of [Me‐14C]choline‐labeled phosphatidylcholines and sphingomyelin taken up by rat Kupffer cells in maintenance culture during interaction with large unilamellar liposomes composed of cholesterol, labeled choline‐phospholipid and phosphatidylserine (molar ration 5:4:1).

With both labeled compounds only small proportions of water‐soluble radioactivity were found to accumulate in the cells and in the culture medium, suggesting limited phospholipid degradation. However, after a lag period of 30 min progressively increasing proportions of cell‐associated liposomal phospholipid were found to be converted to cellular phospholipid, nearly all of which was phosphatidylcholine. This conversion as well as the limited release of watersoluble label from the cells was inhibited by the lysosomotropic agents ammonium chloride and chloroquine.

With [Me‐14C]choline‐labeled lysophosphatidylcholine, label was found to become cell‐associated far in excess of an encapsulated liposomal label, [3H]inulin. Without a lag period virtually all of this was rapidly converted to phosphatidylcholine, a process which was not inhibited by the lysosomotropic agents.

It is concluded that Kupffer cells, after endocytosis of liposomes, degrade the liposomal phospholipids effectively but reutilize the choline moiety for de novo synthesis of cellular phosphatidylcholine.
Protein structure and gene organization of mouse lactate dehydrogenase‐A isozyme
Authors: LI Steven S.‐L. , TIANO Howard F. , FUKASAWA Kayoko M. , YAGI Kiyohito , SHIMIZU Motoyuki , SHARIEF Farida S. , NAKASHIMA Yasutsugu , PAN Yu‐ching E.
Published: March 2005

The complete covalent structure of the 331 amino acids of mouse lactate dehydrogenase (LDH) A4 isozyme has been determined by sequence analyses of both the protein and the genomic DNA. The mouse LDH‐A gene spans a length of at least 7000 bases from the translation initiation codon ATG to the end of the 3′ untranslated region, and it contains six introns that interrupt the protein‐coding sequence. The relationships between the exon‐intron organization of LDH‐A gene and the structural‐functional domains of the protein are discussed.
Isolation and characterization of peroxisomes from the liver of normal untreated rats
Authors: VÖLKL Alfred , FAHIMI H. Dariush
Published: March 2005

The classic method of Leighton et al. [(1968) J. Cell Biol. 37, 482–513] for the isolation of peroxisomes from rat liver involves the use of Triton WR‐1339 which alters the biochemical properties of this organelle and requires the specialized type Beaufay‐rotor which is not easily available. We have employed Metrizamide as the gradient medium and a commercial type vertical rotor to obtain highly purified and structurally well‐preserved peroxisomes from normal untreated animals.

The livers were homogenized in buffered 0.25 M sucrose and a slightly modified ‘light mitochondrial fraction’ was prepared by differential centrifugation. This was loaded on top of a linear Metrizamide gradient (1.12 – 1.26 g/cm3) and subjected to an integrated force of 1.252 × 106× (g× min) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at the density of 1.245 g/cm3. In the isolated fraction 95% of the protein was contributed by peroxisomes, which exhibited a strong activity for cyanide‐insensitive lipid β‐oxidation. The purity of fractions was also confirmed by morphometry, which revealed that 98% of isolated particles consisted of peroxisomes. The latency for catalase was about 90% indicating a high degree of peroxisomal integrity. This corresponded to the low level of extraction of catalase in 3,3′‐diaminobenzidine‐stained filter preparations. The entire procedure took about five hours. Highly purified and structurally well preserved peroxisomes should be useful in further elucidation of the function of this organelle and especially in studies of peroxisomal enzymes with multiple intracellular localizations.
Primer‐independent abortive initiation by wheat‐germ RNA polymerase B (II)
Authors: MOSIG Helmut , SCHÄFFNER Anton R. , SIEBER Heidemarie , HARTMANN Guido R.
Published: March 2005

Highly purified RNA polymerase B (II) from wheat germ catalyses the formation of dinucleoside tetraphosphates from ribonucleoside triphosphates in the absence of an oligonucleotide primer or additional protein factors. The reaction requires bivalent cations such as Mn2+ or Mg2+ and proceeds linearly for several hours. It is strongly inhibited by 1 μg/ml α‐amanitin or 2 μg/ml heparin. The reaction strictly depends on the addition of a specific linear or circular DNA template, such as the plasmid pSmaF or a DNA fragment containing the gene for nopaline dehydrogenase. Bacteriophage T7 D111 DNA has almost no template activity. The start sites for dinucleotide synthesis on the template are limited. With the DNA fragment containing the gene for nopaline dehydrogenase only pppApA and pppApU are synthesised substantially whereas pppUpU is formed only in trace amounts. No significant dinucleotide synthesis is observed with other ribonucleoside triphosphates either singly or in a combination of two. The various regions of the DNA fragment differ distinctly in template activity.
Electron microscopy of the Mo‐Fe‐protein from Azotobacter vinelandii nitrogenase
Authors: TSUPRUN V. L. , MITSOVA I. Z. , BLAZHCHUK I. S. , GVOZDEV R. I. , ORLOVA E. V. , KISELEV N. A.
Published: March 2005

The quaternary structure of the Mo‐Fe‐protein from Azotobacter vinelandii has been studied by electron microscopy. A model of the molecule of the Mo‐Fe‐protein has been proposed: two α subunits are displaced relative to two β subunits along a twofold axis, so the molecule can be characterized by the point‐group pseudosymmetry 222. Computer averaging of the images showed that one of the projections of the molecule could be characterized by twofold rotational symmetry. Micrographs of nitrogenase recombined complex (Mo‐Fe‐protein + Fe‐protein) have been obtained. They showed particles close in size and form to the Mo‐Fe‐protein molecule. Therefore, it has been proposed that the Fe‐protein could be situated in the central cavity of Mo‐Fe‐protein.
Catalytic properties of a human cytomegalovirus‐induced protein kinase
Authors: MICHELSON Susan , TARDY‐PANIT Maryse , BÂRZU Octavian
Published: March 2005

Human cytomegalovirus, a DNA virus whose genome contains a fragment of transforming DNA, induces a threonine‐serine protein kinase having a molecular mass of 68 kDa (p68). p68 was extracted from cells 96–144 h after infection, and immunoprecipitated with a monoclonal antibody (F6b). Antibody‐enzyme complexes were immobilized on heat/formaldehyde‐inactivated Staphylococcus aureus.

The best substrates for p68 were acidic proteins, phosvitin and casein. Glycogen synthase, phosphorylase a and histones were phosphorylated at rates not higher than 1–4% that obtained with phosvitin as substrate. ATP and GTP were equally good substrates of p68. p68 is able to autophosphorylate at the same residues (i.e. threonine and serine) as the protein substrates. Autophosphorylation does not seem to represent an intermediate in substrate phosphorylation.

The protein kinase activity of p68 was not enhanced by cAMP, calcium ions, or polyamines like spermine or spermidine. Only at low Mg2+ concentration spermine enhanced by 68% the rate of casein phosphorylation. Heparin, a potent inhibitor of casein kinase II, inhibits p68 activity too, but ten‐times higher concentrations were required for the same degree of inhibition. Quercetin, a bioflavonoid, acts as a strong inhibitor of p68 protein kinase activity. The inhibitory effect of quercetin was competitive towards the nucleotide substrate (Ki= 2.8 μM), and non‐competitive towards the protein substrate (Ki= 15 μM).
Molecular forms of sphingomyelinase and non‐specific phosphodiesterases in Epstein‐Barr virus‐transformed lymphoid cell lines from Niemann‐Pick disease types A and B
Authors: LEVADE Thierry , SALVAYRE Robert , DOUSTE‐BLAZY Louis
Published: March 2005

Molecular forms of sphingomyelinase and phosphodiesterases from lymphocytes and Epstein‐Barr virus‐transformed lymphoid cell lines were separated by preparative electrofocusing in granulated gels. In either type of cell derived from normal individuals, sphingomyelinase focused as a single peak (pI = 5.60 ± 0.1) while phosphodiesterases hydrolyzing bis(4‐methylumbelliferyl)phosphate and bis(4‐methylumbelliferyl)diphosphate separated into seven and three molecular forms respectively; one of the latter showed sphingomyelinase as well as phosphodiesterase activities. Lymphoid cell lines derived from patients with Niemann‐Pick disease, types A or B, were practically devoid of sphingomyelinase activity; this was not so for the phosphodiesterases which focussed essentially as normal. The protein peak, which in normal cells contained the three activities, had phosphodiesterase but no sphingomyelinase activity in the Niemann‐Pick cells. In normal cells, sphingomyelinase and phosphodiesterase activities of this peak showed different responses to heating and several effectors. These data suggest that in lymphoid cell lines, which are a useful model for studies of Niemann‐Pick disease, sphingomyelinase and phosphodiesterases are subject to separate genetic coding and that the latter activities are not a reliable measure for diagnosing Niemann‐Pick disease.
Glycinin A5A4B3 mRNA: cDNA cloning and nucleotide sequencing of a splitting storage protein subunit of soybean
Authors: MOMMA Takayuki , NEGORO Takaharu , HIRANO Hisashi , MATSUMOTO Akiyo , UDAKA Kyoko , FUKAZAWA Chikafusa
Published: March 2005

The complete nucleotide sequence of a cloned cDNA, designated pGA5A4B3822, corresponding to glycinin A5A4B3 mRNA was determined. Analysis of the cDNA insert revealed that it contained 1899 nucleotides of mRNA sequence with a 5′‐terminal non‐translated region of 31 nucleotides, a signal peptide region corresponding to 23 amino acids, an acidic subunit region (A5) corresponding to 97 amino acids, an acidic subunit region (A4) corresponding to 257 amino acids followed by a basic subunit region (B3) corresponding to 185 amino acids, and a 3′‐terminal non‐translated region of 182 nucleotides. These results show that the glycinin A4 subunit, which is not found to be linked to a basic subunit via a disulfide bond, is synthesized as a full‐sized precursor, i.e. the A5A4B3 subunit complex, from a single mRNA, followed by post‐translational processing to generate an intermediary subunit complex (A5‐B3), covalently linked by a disulfide bond, and the mature A4 subunit, which may associate with the above subunit complex by non‐covalent interactions. From the results obtained by the Chou‐Fasman rules we speculated that the two post‐translational cleavage sites of this subunit precursor might be processed by the same proteolytic enzyme.
Photo‐induced protein cross‐linking to 5S RNA and 28–5.8S RNA within rat‐liver 60S ribosomal subunits
Authors: PALEOLOGUE Anne , REBOUD Anne‐Marie , REBOUD Jean‐Paul
Published: March 2005

Rat liver 60S ribosomal subunits were irradiated with 254‐nm ultraviolet light (1.26 × 104 quanta/subunit), under conditions which preserved their functional activity. Cross‐linked RNA‐protein complexes were recovered after unreacted proteins had been removed by repeated acetic acid extractions. Proteins linked to the whole rRNA, to 5S RNA and to 28–5.8S RNAs were identified by two‐dimensional gel electrophoresis after RNA hydrolysis by ribonucleases T1 and A. Our results showed that numerous proteins interact with rRNAs (at least ten with 28–5.8S RNA, eight with 5S RNA and among these three are common to both) and have been discussed in the light of all the available data.
Solid‐phase synthesis of PYLa and isolation of its natural counterpart, PGLa [PYLa‐(4–24)] from skin secretion of Xenopus laevis
Authors: ANDREU David , ASCHAUER Heinz , KREIL Günther , MERRIFIELD R. Bruce
Published: March 2005

From the nucleotide sequence of clones isolated from a cDNA library constructed from skin of Xenopus laevis, the existence of PYLa, a peptide comprised of 24 amino acids, was predicted. This peptide was synthesized by solid‐phase methods and purified to homogeneity with an overall yield of 61%. The synthetic peptide was used as reference substance to search for its natural counterpart in skin secretion of Xenopus. Two peptides were found which were very similar to PYLa except for the absence of the first three amino acids. These 21‐amino‐acid peptides, termed PGLa, can be generated from PYLa by cleavage after the single arginine residue present in the latter. The two forms of PGLa differ in their retention time on HPLC but have identical amino acid compositions and terminal sequences. Tryptic hydrolysis of synthetic PYLa after the single arginine yields exclusively PGLa with the shorter retention time on HPLC. The chemical difference between the two forms of PGLa is currently not known. The possible biological role of these newly discovered constituents of frog skin secretion is discussed.
Nucleotide sequence, secondary structure and evolution of the 5S ribosomal RNA from five bacterial species
Authors: VANDENBERGHE Antoon , WASSINK Annick , RAEYMAEKERS Peter , DE BAERE Raymond , HUYSMANS Erik , DE WACHTER Rupert
Published: March 2005

The nucleotide sequences of the 5 S ribosomal RNAs of the bacteria Agrobacterium tumefaciens, Alcaligenes faecalis, Pseudomonas cepacia, Aquaspirillum serpens and Acinetobacter calcoaceticus have been determined. The sequences fit in a generally accepted model for 5 S RNA secondary structure. However, a closer comparative examination of these and other bacterial 5 S RNA primary structures reveals the potential of additional base pairing and of multiple equilibria between a set of slightly different alternative secondary structures in one area of the molecule. The phylogenetic position of the examined bacteria is derived from a 5 S RNA sequence alignment by a clustering method and compared with the position derived on the basis of 16 S ribosomal RNA oligonucleotide catalogs.
Chemical modification of methionine residues of the phosphatidylcholine transfer protein from bovine liver. A spin‐label study
Authors: MEGLI Francesco M. , van LOON Douwe , BARBUTI Angela A. , QUAGLIARIELLO Ernesto , WIRTZ Karel W. A.
Published: March 2005

The role of methionine residues in the interaction of the phosphatidylcholine transfer protein from bovine liver with phospholipid vesicles was investigated by specific modification of these residues with iodoacetamide. The modified protein was digested with cyanogen bromide in order to determine which methionine residues had become resistant to this cleavage. Automated Edman degradation on the digest indicated that after 72 h of reaction, Met‐1 was modified for 80%, Met‐73 for 50%, Met‐109 for 20%, whilst Met‐173 and Met‐203 were found to be unmodified.

This distinct modification did not result in any loss of phosphatidylcholine transfer activity. The interaction of the phosphatidylcholine transfer protein with phospholipid vesicles was investigated by making use of electron spin resonance spectroscopy. The interaction of unmodified protein with vesicles composed of phos‐phatidylcholine/phosphatidic acid/spin‐labeled phosphatidylethanolamine (79:16:5, mol%) or composed of phosphatidylserine/spin‐labeled phosphatidylethanolamine (95:5, mol%), gave an increase of about 50% in the rotation correlation time.

A similar increase was observed with the modified protein. This interaction was further investigated by labeling Met‐1 and Met‐73 in the transfer protein with iodoacetamidoproxyl spin‐label.

Spin‐labeling did not inactivate the transfer protein. In addition, the electron spin resonance spectra of the spin‐labeled protein were not affected upon addition of vesicles composed of phosphatidylcholine/phosphatidic acid (80:20, mol%). These experiments strongly suggest that Met‐1 and Met‐73 are not part of the site that interacts with the membrane.
Quantitative and mechanistic aspects of the hydroperoxide‐induced release of Ca2+ from rat liver mitochondria
Authors: FREI Balz , WINTERHALTER Kaspar H. , RICHTER Christoph
Published: March 2005

We have previously demonstrated in rat liver mitochondria a hydroperoxide‐induced hydrolysis of pyridine nucleotides and release of Ca2+ [Lötscher, H. R., Winterhalter, K. H., Carafoli, E. & Richter, C. (1979) Proc. Natl Acad. Sci. USA 76, 4340–4344, and Lötscher, H. R., Winterhalter, K. H., Carafoli, E. & Richter, C. (1980) J. Biol. Chem. 255, 9325–9330]. Here we investigate pyridine nucleotide hydrolysis and Ca2+ release under conditions of minimized Ca2+ cycling and with smaller Ca2+ loads. The extent of pyridine nucleotide hydrolysis, measured by pyridine‐nucleotide‐derived nicotinamide release from intact mitochondria, and the Ca2+ release rate show a very similar sigmoidal dependence on the mitochondrial Ca2+ load. The hydrolysis of oxidized pyridine nucleotides is limited under non‐cycling conditions. Whereas pyridine nucleotide hydrolysis as measured by nicotinamide release is extensive, net loss of mitochondrial pyridine nucleotides is observed only at relatively high Ca2+ loads. Our results indicate the ability of mitochondria to resynthesize pyridine nucleotides after hydrolysis. Neither a decrease of reduced, nor an increase of oxidized, mitochondrial glutathione favour Ca2+ release. From these and previous findings it is concluded that the hydroperoxide‐induced Ca2+ release is triggered by a factor which is distal to the oxidation of mitochondrial pyridine nucleotides. Ca2+ release is stimulated when the movement of protons across the inner mitochondrial membrane is facilitated, giving evidence for the operation of the hydroperoxide‐induced release pathway as a Ca2+/H+ antiport.
Mechanism of carboxypeptidase‐Y‐catalyzed reaction deduced from a pressure‐dependence study
Authors: FUKUDA Mitsuhiro , KUNUGI Shigeru
Published: March 2005

The activation volumes for kcat of the carboxypeptidase‐Y‐catalyzed hydrolysis of ester substrates were slightly negative (−1 to −4 ml/mol), while those for peptide and depsi‐peptide analog were highly positive (+ 10 to + 27 ml/mol). These values and the contrasting pH dependences of these two groups of the substrates are explained by a mechanism involving three ionic states of the enzyme and the second stable intermediate (acyl‐enzyme). Esters are mostly rate‐controlled by the deacylation step and peptides are controlled by both the acylation and the deacylation steps. Pressure increase induced a partial shift of the rate‐determining step. Reaction volumes for K−1m of peptide and depsi‐peptide analog showed large and positive values (+16 to + 29 ml/mol) which reflects the electrostatic interaction in the substrate recognition by this enzyme.
Prokaryotic triterpenoids
Authors: ZUNDEL Magali , ROHMER Michel
Published: March 2005

The incorporation of L‐[methyl‐3H,14C]methionine or L‐(methyl‐2H3)methionine into 2β‐methyldiplopterol of Methylobacterium organophilum and various 3β‐methylhopanoids of Acetobacter pasteurianus ssp. pasteurianus showed that all three hydrogen atoms of the transferred methyl group are retained in the triterpenoids. These methylations are compatible with a methylation substrate such as a Δ2‐hopanoid in the case of the 2β‐methylhopanoid biosynthesis and of a Δ2‐hopanoid or squalene in the case of the formation of 3β‐methylhopanoids. The intervention of intermediates possessing an exomethylene group or a cyclopropane ring is excluded.
Purification and characterization of glutamate decarboxylase from Solanum tuberosum
Authors: SATYANARAYAN Vaduvatha , NAIR P. Madhusudanan
Published: March 2005

Glutamate decarboxylase has been purified from potato tubers. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Gel filtration on Sephadex G‐200 gave a relative molecular mass Mr, of 91000 for the native enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a subunit Mr of 43000. Thus the enzyme appears to be a dimer of identical subunits. It has 2 mol pyridoxal 5′‐phosphate/mol protein, which could not be removed by exhaustive dialysis or gel filtration on Sephadex G‐25. The enzyme has an absorpion maximum at 370 nm in sodium phosphate buffer, pH 5.8. Reduction of the enzyme with sodium borohydride abolished the absorption maximum at 370 nm with attendant loss of catalytic activity. The enzyme exhibited pH‐dependent spectral changes. The enzyme was specific for L‐glutamate and could not decarboxylate other amino acids tested. The enzyme was maximally active at pH 5.8 and a temperature of 37°C. Isoelectric focussing gave a pI of 4.7 Km values for L‐glutamate and pyridoxal 5′‐phosphate were 5.6 mM and 2 μM respectively. Thiol‐directed reagents and heavy metal ions inhibited the enzyme, indicating that an –SH group is required for activity. The nature of the functional groups at the active site of the enzyme was inferred from competitive inhibition studies. L‐Glutamate promoted inactivation of the enzyme caused by decarboxylation‐dependent transamination was demonstrated. The characteristics of potato enzyme were compared with enzyme from other sources.
Diastereomers of adenosine 3′,5′‐monothionophosphate (cAMP[S] antagonize the activation of cGMP‐dependent protein kinase
Authors: HOFMANN Franz , GENSHEIMER Hans‐Peter , LANDGRAF Wolfgang , HULLIN Roger , JASTORFF Bernd
Published: March 2005

cGMP‐dependent protein kinase contains four cGMP‐binding sites which are homologeous to the four cAMP‐binding sites of cAMP‐dependent protein kinase. The interaction of the diastereomers of adenosine 3′,5′‐thionophosphate, (PS)‐cAMP[S] and (PR)‐cAMP[S], with cGMP‐dependent protein kinase has been studied.

1. Autophosphorylation of cGMP‐dependent protein kinase is stimulated by cAMP and (PS)‐cAMP[S] with apparent KA values of 7 μM and 94 μM, respectively.

2. cAMP‐stimulated autophosphorylation is inhibited competitively by (PR)‐cAMP[S] with a Ki value of 15 μM.

3. The phosphorylation of the peptide substrate (Leu‐Arg‐Arg‐Ala‐Ser‐Leu‐Gly) is stimulated by cGMP (approx. KA 1 μM) and cAMP (approx. KA 98 μM) but neither by the (PR) nor (PS) stereomer of cAMP[S].

4. (PR)‐cAMP[S] and (PS)‐cAMP[S] inhibit competitively cAMP‐ or cGMP‐stimulated phosphorylation of the peptide substrate with Ki values of 52 μM and 73 μM, respectively.

5. (PS)‐cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme.

6. Binding of [3H]cGMP to cGMP‐dependent protein kinase is inhibited by (PS)‐cAMP[S] and (PR)‐cAMP[S] with IC50 values of 200 μM and 15 μM, respectively.

7. These results show that both diasteromers of cAMP[S] bind to cGMP‐dependent protein kinase. (PR)‐cAMP[S] has properties of a pure antagonist whereas (PS)‐cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP‐binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.
Demonstration of a bacteriophage receptor site on the Escherichia coli K12 outer‐membrane protein OmpC by the use of a protease
Authors: MORONA Renato , TOMMASSEN Jan , HENNING Ulf
Published: March 2005

The Escherichia coli K12 outer‐membrane proteins OmpA, OmpC, OmpF, PhoE, and LamB (all of transmembrane nature) can serve as phage receptors. We have shown previously that one OmpA‐specific phage, Ox2, can give rise to the host range mutants Ox2h10 and Ox2h12, with the latter being derived from the former [Morona. R. & Henning, U. (1984) J. Bacteriol. 159, 579–582]. Unlike Ox2, both host range phages can use the OmpA and OmpC proteins as receptors and Ox2h12 is better adapted to the OmpC protein than Ox2h10. In a search for the site(s) of OmpC protein involved in phage recognition, it was found that proteinase K is able to cleave all of the proteins mentioned above. OmpC protein (Mr= 38306) could be cleaved from outside the cell by proteinase K resulting in two fragments of Mr∼ 21000 and Mr∼ 17500. The use of OmpC‐PhoE hybrid proteins allowed us to assign the ∼ 21000‐Mr fragment to the CO2H‐terminal moiety of the protein. Proteinase K treatment of intact cells abolished their activity to neutralize the OmpC‐specific phage Tulb and reduced this ability towards phage Ox2h12. The OmpA, OmpF, PhoE and LamB proteins were cleaved by the protease not in intact cells but only when acting on cell envelopes.

The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150–178 of the 346‐residue protein, which shows homology to two regions of the OmpA protein which are involved in its phage receptor site (loc. cit.). These areas also exhibit some homology to a region of the LamB protein which is thought to be part of this protein's receptor site [Charbit et al. (1984) J. Mol. Biol. 175, 395–401]. This suggests that there is a common denominator for proteinaceous phage receptor site because the LamB‐specific phage λ and phage Tulb are of completely different nature. We conclude that the region of the OmpC protein in question is cell‐surface‐exposed and acts as a phage receptor site.
Role of plasma albumin in renal elimination of a mercapturic acid
Authors: OKAJIMA Kenji , INOUE Masayasu , ITOH Kazunobu , NAGASE Sumi , MORINO Yoshimasa
Published: March 2005

Biosynthesis of N‐acetylcysteine S‐conjugates of xenobiotics (mercapturic acids) occurs via interogan metabolism and the renal transtubular transport system plays an important role in elimination of the final metabolites from the organism. To assess the behavior of a mercapturic acid in the circulation, plasma clearance of radioactive S‐benzyl‐N‐acetylcysteine and its interaction with plasma proteins were studied in normal and mutant analbuminemic rats (NAR). Intravenously injected S‐benzyl‐N‐acetylcysteine rapidly disappeared from the circulation both in NAR and normal animals. However, its plasma clearance was significantly higher in NAR (45.7 ml kg−1 min−1) than in normal rats (25.2 ml kg−1). Ultrafiltration analysis revealed that 18.4% and 80.1% of the mercapturate bound to plasma protein(s) from NAR and normal rats, respectively, at 50 μM ligand concentration. The mercapturic acid bound to plasma albumin with an association constant of 2.24 × 105 M−1 and the number of binding sites was 1.18/mol albumin. The binding was competitively inhibited by probenecid and L‐tryptophan. Concomitant administration of this mercapturic acid with equimolar amounts of albumin resulted in a marked decrease in the plasma clearance (26.2 ml kg−1 min−1) and an increase in the urinary secretion of this ligand in NAR. 30 min after injection of the mercapturic acid (10 μmol/kg body weight), 27.3% and 60.4% of the injected dose was recovered from urine and kidneys of NAR and normal rats respectively. About 41% of the dose was recovered in NAR urine when the ligand was injected bound to an equimolar amount of albumin. These results suggested that albumin is important for the renal accumulation and urinary elimination of the circulating mercapturic acid.
Bundling of microtubules by glyceraldehyde‐3‐phosphate dehydrogenase and its modulation by ATP
Authors: HUITOREL Philippe , PANTALONI Dominique
Published: March 2005

Glyceraldehyde‐3‐phosphate dehydrogenase from different origins (brain, muscle, erythrocytes) binds to microtubules polymerized from pure brain tubulin and causes bundle formation in vitro. ATP is shown to dissociate these bundles into individual microtubules, while the dehydrogenase is not displaced from the polymers by this nucleotide. ATP can be replaced by adenosine 5′‐(ß,γ‐imido]triphosphate, a nonhydrolyzable analog of ATP. These data are interpreted in terms of dissociation of the glyceraldehyde‐3‐phosphate dehydrogenase tetramer into dimers by ATP. The enzyme is also efficiently purified by a tubulin‐Sepharose affinity chromatography.
Identification of the calmodulin‐binding components in bovine lens plasma membranes
Authors: LOUIS Charles F. , JOHNSON Ross , TURNQUIST Janet
Published: March 2005

Lens membranes, purified from calf lenses, have been labeled by covalent cross‐linking to membrane‐bound 125I‐calmodulin with dithiobis(succinimidyl propionate). Electrophoretic analysis in sodium dodecyl sulfate demonstrated two major 125I‐containing products of Mr= 49000 and 36000. That the formation of these two components was specifically inhibited by unlabeled calmodulin, or calmodulin antagonists, would indicate that the formation of these components was calmodulin‐specific. The size of these two 125I‐labeled components was unchanged over a range of 125I‐calmodulin or dithiobis(succinimidyl propionate) concentrations indicating that they represent 1:1 complexes between 125I‐calmodulin (Mr= 17000) and Mr‐32000 and Mr‐19000 lens membrane components respectively. Although formation of both cross‐linked components exhibited an absolute dependence on Mg2+, the autoradiographic intensity of these components was enhanced when Ca2+ was included with Mg2+ during the cross‐linking reaction. Labeling was maximal in 10 mM MgCl2 and approximately 1 μM Ca2+. Treatment of lens membranes with chymotrypsin resulted in the cleavage of MP26 (the major lens membrane protein), with the appearance of a major proteoloytic fragment of Mr= 22000. This proteolysis was not associated with any significant change in either the size or amount of the 125I‐calmodulin‐labeled membrane components. These results suggest that calmodulin interacts with two membrane proteins, but not significantly with MP26, in the intact lens cell membrane. Our results indicate the need to maintain caution in interpreting direct calcium plus calmodulin effects on MP26 and lens cell junctions.
Cloning and nucleotide sequence of the murine interleukin‐3 gene
Authors: CAMPBELL Hugh D. , YMER Sanie , FUNG Ming‐chiu , YOUNG Ian G.
Published: March 2005

Southern hybridization analysis using a probe derived from a murine interleukin‐3 (IL‐3) cDNA clone revealed the presence of a single IL‐3 gene in the haploid murine genome. An 8600‐base‐pair (8.6‐kb) murine genomic EcoRI fragment containing the IL‐3 gene was isolated by screening a library of size‐fractionated genomic EcoRI fragments cloned in λgt WES. λB. The nucleotide sequence of a 3.5‐kb region of the cloned DNA encompassing the IL‐3 gene was determined. The gene contains four introns of 96, 993, 135 and 122 base pairs (bp), located within the coding region. The large intron contains 12 copies of a 14–15‐bp tandem repeating sequence which resemles a human cellular homologue of a BKV enhancer sequence. The nucleotide sequence of the exons agrees exactly with that of an IL‐3 cDNA cloned from WEHI‐3, a tumorigenic cell line which over‐produces IL‐3, establishing that the unprocessed primary structure of IL‐3 is identical in WEHI‐3 and in BALB/c mice. Southern hybridization has revealed genomic alteration in the vicinity of the IL‐3 gene in WEHI‐3 cells.
Nucleotide sequence of cucumber mosaic virus RNA 1
Authors: REZAIAN M. Ali , WILLIAMS Rhys H. V. , SYMONS Robert H.
Published: March 2005

The nucleotide sequence of the 3389 residues of RNA 1 (Mr1.15 × 106) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy‐chain‐termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5′ end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98–100), which is the start of a single long openreading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3′ end of RNA 1. In addition to the conserved 3′ region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5′ leader sequences, a 12‐nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12‐nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr‐126000 protein.