bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial reassembly of active 60S ribosomal subunits from rat liver following treatment with dimethylmaleic anhydride
Authors: CONQUET François , LAVERGNE Jean‐Pierre , PALEOLOGUE Anne , REBOUD Jean‐Paul , REBOUD Anne‐Marie
Published: March 2005

Rat liver 60S ribosomal subunits were treated with dimethylmaleic anhydride, a reagent for protein amino groups, at a 1/15 000 mol/mol ratio. This caused the dissociation of specific proteins, which were separated from the 56S residual core particles by centrifugation and identified by two‐dimensional gel electrophoresis. The core particles lacking 30% of the total proteins retained most of the initial activity measured by the puromycin reaction but only small percentages of activities measured by polyphenylalanine synthesis, elongation‐factor‐2(EF‐2)‐dependent GTP hydrolysis and EF‐2‐mediated GDP binding. Upon reconstitution, the complementary amount of split proteins was incorporated into ribosomal particles, which had almost the same catalytic activities and biophysical properties (density, sedimentation coefficient and capability to reassociate to 40S subunits) as the original subunits.
Expression of bovine pancreatic ribonuclease A in Escherichia coli
Authors: NAMBIAR Krishnan P. , STACKHOUSE Joseph , PRESNELL Scott R. , BENNER Steven A.
Published: March 2005

A synthetic gene for bovine pancreatic ribonuclease A (RNase A) has been expressed in Escherichia coli as a fusion protein with β‐galactosidase linked by the tetrapeptide Ile‐Glu‐Gly‐Arg. RNase A was cleaved from the fusion using factor Xa, and the resulting product prufied and reconstituted. The isolated RNase A was chromatographically, catalytically, and immunologically indentical with authentic RNase A. This work argues that the method suggested by Nagai and Thogersen [Nagai, K. & Thogersen, H. C. (1984) Nature (Lond.) 309, 810–812] for releasing fusion proteins is quite general, even when applied to a particularly complicated expression problem. The procedure here makes RNase A available for the first time as a model for studying structure‐function relationships in proteins using site‐directed mutagenesis.
Nucleotide sequence of the gene for the peptidoglycan‐associated lipoprotein of Escherichia coli K12
Authors: CHEN Robert , HENNING Ulf
Published: March 2005

During attempts to clone the gene for the Escherichia coli outer membrane protein III another gene was recovered. Its nucleotide sequence was determined and the deduced amino acid sequence showed that the gene does not encode protein III. It codes for a 173‐residue polypeptide; 21 NH2‐terminal residues are typical for a signal peptide. The sequence around the putative site (Ala‐Cys) for removing this peptide, Ala‐Ile‐Ala‐Ala‐Cys‐Ser‐Ser‐Asn, is highly homologous to that of the major cell envelope lipoprotein (Braun lipoprotein) surrounding its processing site; it is also homologous to the consensus pentapeptide Leu‐Leu‐Ala‐Gly‐Cys present in other lipoproteins of gram‐negative bacteria. It could be shown that the gene expresses a lipoprotein with all the properties, including the amino acid composition, of the peptidoglycan‐associated lipoprotein (PAL) [Mizuno, T. (1979) J. Biochem. (Tokyo) 86, 991–1000]. Therefore, the cloned gene is the pal gene. The protein does not contain hydrophobic regions which would serve as a membrane anchor. Tandemly repeated amino acid sequences exist at and near the NH2‐terminus of the mature protein which are homologous to such repeats in the Braun lipoprotein, suggesting a common origin of this part of the two proteins. Attempts to place a transposon into the pal gene were unsuccessful. Hence the complete absence of the protein may be lethal and its function remains unknown.
Subunit II of yeast QH2:cytochrome‐c oxidoreductase
Authors: OUDSHOORN Peter , VAN STEEG Harry , SWINKELS Bart W. , SCHOPPINK Peter , GRIVELL Leslie A.
Published: March 2005

The nucleotide sequence of a 2.5 × 103‐base segment of yeast nuclear DNA, containing the structural gene for the 40‐kDa subunit II of the ubiquinol:cytochrome‐c oxidoreductase, has been determined. The region contains only one single reading frame of length sufficient to encode a protein of the size of subunit II. The mature protein is predicted to have a length of 352 amino acids, with a molecular mass of 38 714 Da. It is predominantly hydrophilic, with an overall polarity of 45%. Comparison of the sequence of the reading frame with that derived from direct sequence analysis of the N terminus of the mature 40‐kDa protein shows that subunit II is synthesized as a longer precursor and shows that the extension is N‐terminal. The presequence is 16 amino acids long and it contains a number of positively charged residues and lacks acidic ones. It is also rich in neutral, polar amino acids. S1 nuclease protection analysis of DNA · RNA hybrids identifies two major and one minor transcript of the gene, whose 5′ termini map approximately 55, 65 and 75 nucleotides upstream of the initiation codon. Sequences 5′ of these termini lack obvious homology to the regulatory sequences of other imported mitochondrial proteins, whose synthesis is controlled by oxygen and by catabolite repression. A mutant lacking a functional subunit II gene has been constructed by a one‐step gene‐disruption procedure. This mutant grows only slowly on glycerol and still displays a low level of QH2: cytochrome‐c oxidoreductase activity (approx. 5% of that of wild type). The implications of this finding for the possible role of subunit II in the complex are discussed.
The complete amino acid sequence of rubredoxin from the green phototrophic bacterium Chlorobium thiosulphatophilum strain PM
Authors: WOOLLEY Kevin J. , MEYER Terry E.
Published: March 2005

A complete amino acid sequence for the rubredoxin from the photosynthetic bacterium Chlorobium thiosulphatophilum is proposed. The sequence, a single polypeptide chain of 53 amino acids, was deduced from the sequences of peptides obtained by chymotryptic, tryptic, thermolytic or mild acid digestion.

The rubredoxin shows a high degree of sequence homology with rubredoxins from non‐photosynthetic bacteria, and the evolutionary implications of this are considered.
Regulation of synthesis and cell content of the low‐density‐lipoprotein receptor protein in cultured fibroblasts from normal and familial hypercholesterolaemic subjects
Authors: KNIGHT Brian L. , PATEL Dilip D. , SOUTAR Anne K.
Published: March 2005

Incorporation of [35S]methionine into low‐density‐lipoprotein (LDL) receptors by normal fibroblasts and those from a homozygous familial hypercholesterolaemic (FH) subject who produced defective but immunoprecipitable receptor proteins of normal size, was compared with the ability of the cells to bind LDL and their content of LDL receptor protein determined using a double‐antibody radioimmunoassay. The FH cells produced precursor protein with a longer half‐life (3–4 h) than normal cells (40 min), most of which was eventually processed to a mature form of the receptor. Total receptor half‐life was similar to normal (approx. 12 h) and LDL binding about 20% of normal.

Incubation of normal fibroblasts with lipoprotein‐deficient serum (LPDS) led to an increase in the amount of LDL receptor protein in the cells which was closely followed by the increase in their ability to bind LDL. Receptor synthesis increased rapidly at first, but then fell by more than 60% before remaining constant. The peak of synthesis coincided with the greatest rate of increase in receptor content. At equilibrium in LPDS receptor synthesis, LDL binding and receptor protein content were all approximately 3.3‐fold higher than in cells maintained in 10% foetal calf serum (FCS). The FH cells also responded to LPDS with a rise and fall in the rate of receptor synthesis. They did not compensate for their inefficiency in producing active receptors with an increase in total receptor content. In LPDS, peak synthesis and maximum receptor content of the FH cells were similar to normal. In FCS receptor synthesis and content were well below maximum so that they did not fully employ even the low capacity for LDL uptake of which they were capable. With both types of cell, inhibition of mevalonic acid and cholesterol synthesis with compactin delayed, but did not prevent the secondary fall in the rate of receptor synthesis, again suggesting a regulatory role for some factor not directly related to cholesterol metabolism.
Hormonal regulation of the rate of the glycogen/glucose‐1‐phosphate cycle in skeletal muscle
Authors: CHALLISS R. A. John , CRABTREE Bernard , NEWSHOLME Eric A.
Published: March 2005

A method is presented which allows simultaneous estimation of rates of glycogen synthesis and glycogenolysis in an isolated incubated skeletal muscle, thus allowing measurement of the glycogen/glucose‐1‐phosphate substrate cycling rate. In the absence of hormonal additions, the measured rates of glycogen synthesis and breakdown were similar [respectively, 0.54 ± 0.12 (8) and 0.74 ± 0.10 (8) μmol glucosyl equiv. h−1 (g tissue)−1]. Incremental addition of insulin stimulated glycogen synthesis up to threefold and reduced glycogenolysis by about sevenfold; the half‐maximally effective concentration of insulin on both processes was about 100 μU/ml (0.7 nM). Incremental addition of adrenaline (in the presence of 1 mU insulin/ml) caused a dramatic increase in the glycogenolytic rate (about 15‐fold), but a much less marked inhibition of glycogen synthetic rate. In addition to hormonal manipulation of the muscle preparation in vitro, the effects of cold exposure, the hyperthyroid state, a single exercise bout and exercise‐training of animals in vivo on the rates of glycogen synthesis and breakdown in the isolated incubated muscle preparation have been investigated. Significant changes in measured glycogen synthesis, breakdown and glycogen/glucose‐1‐phosphate cycling have been observed, both under basal conditions and in response to hormonal additions in vitro. The results are discussed with respect to the possible physiological importance of this substrate cycle.
Receptor‐mediated endocytosis of apamin by liver cells
Authors: STRONG Peter N. , EVANS W. Howard
Published: March 2005

The binding and uptake of the bee venom toxin apamin, by guinea‐pig and rat liver were studied.

Guinea‐pig liver plasma membranes contain inhibitable, high‐affinity binding sites for [125I]monoiodoapamin: Kd= 12.6±0.8 pM (SE); Bmax= 4.2±0.2 fmol/mg protein. No binding sites for [125I]monoiodoapamin on rat liver plasma membranes were detected in agreement with the absence of a physiological response to the toxin by rat hepatocytes.

[125I]Monoiodoapamin, injected into the portal vein of guinea‐pigs, was recovered in an undegraded form in a liver endosome fraction. The uptake of [125I]monoiodoapamin by rat livers was less than 4% of that taken up by guinea‐pig livers and there was little evidence of radiolabelled toxin appearing in isolated rat endocytic vesicles.

Inhibitable, high‐affinity binding sites for [125I]monoiodoapamin were also identified on isolated guinea‐pig liver endosomal membranes; Kd= 10.6±3.3 pM; Bmax= 2.5±0.6 fmol/mg protein. No inhibitable apamin binding sites were detected on rat endosomal membranes.

Plasma membranes and endosomal membranes isolated from guinea‐pig liver showed a similar spectrum of polypeptides to that previously reported for plasma membranes and endosomal membranes isolated from rat liver. The enzymatic composition of guinea‐pig endosomes was also similar to that previously reported for rat endosomes.

The results indicate that apamin was internalised by receptor‐mediated endocytosis by guinea‐pig liver cells in an analogous manner to that already shown for a variety of endogenous ligands.
Formation of D‐Phe‐Pro‐Val‐cyclo‐Orn by gramicidin S synthetase in the absence of L‐leucine
Authors: VATER Joachim , SCHLUMBOHM Wilhelm , PALACZ Zbigniew , SALNIKOW Johann , GADOW André , KLEINKAUF Horst
Published: March 2005

The preparation of both enzymes of gramicidin S synthetase was efficiently improved by introduction of the fast protein liquid chromatography technique. High‐resolution anion‐exchange chromatography on Pharmacia Mono Q HR 5/5 was used as the final purification step. D‐Phe‐Pro‐Val‐cyclo‐Orn was obtained as a product of the multienzyme by omission of L‐leucine from the complete bioassay mixture. This tetrapeptide was formed by cyclization of the C‐terminal ornithine to 3‐amino‐2‐piperidone. It was identified and characterized by chromatographic and spectroscopic procedures using chemically synthesized reference compounds.
Recombinant‐DNA‐derived bovine growth hormone from Escherichia coli
Authors: LANGLEY Keith E. , BERG Thor F. , STRICKLAND Thomas W. , FENTON Dennis M. , BOONE Thomas C. , WYPYCH Jette
Published: March 2005

The isolation of bacterially synthesized, recombinant‐DNA‐derived, bovine growth hormone (r‐bGH) with native structure is described. The r‐bGH is found in insoluble form, in a pellet fraction, after cell breakage and centrifugation. Cell envelope components (protein, lipid, endotoxin) and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction. We demonstrate that the r‐bGH is largely reduced until solubilized using 6 M guanidine/HCl. Air oxidation is then carried out, in the presence of the guanidine/HCl. The oxidation results in a mixture of about one‐third disulfide‐linked oligomers and two‐thirds oxidized monomer. The latter may include some incorrectly oxidized material, but appears to be mostly correctly oxidized. The oxidized monomer is isolated by gel filtration in the presence of guanidine/HCl. Subsequent guanidine/HCl removal leads to refolded, oxidized r‐bGH. All steps in the procedure, in particular the oxidation and refolding steps, can be carried out at relatively high protein concentrations.
Conformations of yeast α‐mating factor and analog peptides as bound to phospholipid bilayer
Authors: WAKAMATSU Kaori , OKADA Akihiko , MIYAZAWA Tatsuo , MASUI Yoshihiro , SAKAKIBARA Shumpei , HIGASHIJIMA Tsutomu
Published: March 2005

The transferred nuclear Overhauser effects of yeast α‐mating factor [(1–13)peptide] in the presence of various spin‐labeled phosphatidylcholines in small unilamellar vesicles of perdeuterated phosphatidylcholine have been analyzed. From the analysis of the quenching effect by spin‐labels, the depth of amino acid side chains of the mating factor in phospholipid bilayer has been elucidated. The Leu4 and Leu6 residues are buried deeply in the apolar region of the phospholipid bilayer while the hydrophilic residues such as Gln5 and Lys7 are in the shallow region of the bilayer. The interaction of the side chains of Trp1 and Trp3 residues of α‐mating factor with the hydrophobic interior of the bilayer contributes to the binding of this peptide with the phosphatidylcholine bilayer. The conformation of des‐Trp1‐α‐mating‐factor [(2–13)peptide] in the membrane‐bound state has been found to be similar to that of (1–13)peptide from the analysis of transferred nuclear Overhauser effects in the presence of mixed vesicles of perdeuterated phosphatidylcholine and perdeuterated phosphatidylserine. The incorporation of this acidic phospholipid in the vesicle remarkably enhances the binding of (1–13)peptide and analog peptides. However, such modifications that weaken the interaction with phospholipid bilayer (deletion of Trp1 and substitution of Trp3 by Gly or Ala) appreciably lower the physiological activity. Transferred nuclear Overhauser effect analyses have also been made of [DHis2]peptide, [DLeu6]peptide and [DLys7]peptide in the presence of the vesicles of perdeuterated phosphatidylcholine. The main‐chain conformations of these three analogs in the membrane‐bound state have been found to be similar to that of (1–13)peptide, although the side‐chain conformations of the D‐amino acid residues are naturally different from those of the L‐amino acid ones. Thus, the physiological activities of the (1–13)peptide and a variety of analog peptides are found to correlate with the affinities to the phosphatidylcholine/phosphatidylserine membrane and with the molecular conformations in the membrane‐bound state.
Proteinase yscD mutants of yeast
Authors: GARCÍA‐ALVAREZ Nieves , TEICHERT Ulrich , WOLF Dieter H.
Published: March 2005

Mutants of the yeast Saccharomyces cerevisiae, devoid of proteinase yscD activity, were isolated by screening for the inability of mutagenized cells to hydrolyze Ac‐Ala‐Ala‐Pro‐Ala‐β‐naphthylamide in situ. One of the selected mutants bears a thermolabile activity pointing to the gene called PRD1 as being the structural gene for proteinase yscD. All mutants isolated fell into one complementation group. The defect segregates 2:2 in meiotic tetrads indicating a single gene mutation, which was shown to be recessive. Diploids heterozygous for PRD1 display gene dosage. The absence of proteinase yscD did not affect mitotic growth under rich or poor growth conditions, neither mating nor ascopore formation. Also growth of mutant cells after a nutritional shift‐down was not altered. Inactivation of enzymes tested which are subject to carbon‐catabolite inactivation, a process proposed to be of proteolytic nature, is not affected by the absence of proteinase yscD. Protein degradation rates in growing cells, in cells under conditions of differentiation or heat shock, showed no obvious alteration in the absence of proteinase yscD activity. Also inactivation of α‐factor pheromone was not affected by proteinase yscD absence. Normal growth of mutant cells on glycerol indicates that the enzyme is not involved in any vital event in mitochondrial biogenesis.
Self‐association and protonation of adenosine 5′‐monophosphate in comparison with its 2′‐ and 3′‐analogues and tubercidin 5′‐monophosphate (7‐deaza‐AMP)
Authors: TRIBOLET Roger , SIGEL Helmut
Published: March 2005

The concentration dependence of the chemical shifts of the protons H‐2, H‐8 and H‐1′ for 2′‐, 3′‐ and 5′‐AMP2− and of the protons H‐2, H‐7, H‐8 and H‐1′ for tubercidin 5′‐monophosphate (= 7‐deaza‐AMP2−; TuMP2−) has been measured in D2O at 27°C to elucidate the self‐association of the nucleoside monophosphates (NMPs). The results are consistent with the isodesmic model of indefinite non‐cooperative stacking; the association constants for all four NMPs are very similar: K∼ 2 M−1. These 1H‐NMR measurements and those on the dependence of the chemical shifts on the pD of the solutions indicate that the NMP2− species exist predominately in the anti conformation Comparison of the shift data for 5′‐TuMP and 5′‐AMP shows that no hydrogen bonding between N‐7 and ‐PO3H− occurs; hence, the previously observed and confirmed ‘wrongway’ chemical shift [Martin, R. B. (1985) Acc. Chem. Res 18, 32] connected with the deprotonation of the ‐PO3H− group most probably results from the anisotropic properties of the phosphate group which is in the anti conformation close to N‐7. From the dependence between the chemical shift and the‐pD of the solutions the acidity constants were calculated for the four protonated NMPs, and for adenosine and D‐ribose 5′‐monophosphate. The measurements also allow an estimation of the first acidity constant of H3(5′‐AMP)+ (pKDD3(AMP)= 0.9 and pKHH3(AMP)= 0.4). The values for pKHH2(NMP) and pKHH(NMP) were also determined from potentiometric pH titrations in aqueous solution (I= 0.1 M, NaNO3; 25°C). The agreement of the results obtained by the two methods is excellent. The position of the phosphate group at the ribose moiety and the presence of N‐7 in the base moiety influence somewhat the acid‐base properties of the mentioned NMPs. Measurements with 5′‐AMP in 50% (v/v) aqueous dioxane show that lowering of the solvent polarity facilitates removal of the proton from the H+(N‐1) site while the ‐PO2−3 group becomes more basic; this increases the pH range in which the monoprotonated H(5′‐AMP)− species is stable and which is now also extended into the physiological pH region. Some consequences of this observation for biological systems are indicated.
The two fast‐reacting thiols of 3‐phosphoglycerate kinase are structurally juxtaposed
Authors: VAS Mária , CSANÁDY György
Published: March 2005

The two fast‐reacting thiol groups of pig muscle 3‐phosphoglycerate kinase can be simultaneously blocked by one mole equivalent of bifunctional reagent: either mercuric chloride (HgCl2) or 1,4‐bis(bromomercuri)butane. The reactions are accompained by an enzyme activity loss of about 50–70% and 60–80% with mercuric chloride and 1,4‐bis(bromomercuri)butane respectively. Removal of either of the reagents with excess cysteine leads to the recovery of at least 70–90% of the original enzymic activity.

Gel chromatographic analysis revealed no change in the molecular mass of the enzyme modified with mercuric chloride, while an increase of about 30% of the apparent molecular mass was observed after the reaction with 1,4‐bis(bromomercuri)butane. Since no dimer formation could be detected by independent crosslinking, the increase of the apparent molecular mass is probably due to modification causing protein conformational change. The results strongly suggest that the fast‐reacting thiols are intramolecúlarly connected by either of the above bifunctional reagents. In the light of the known structural data on the enzyme, it may follow that the two fast‐reacting thiols belong to the two sequentially neighbouring cysteinyl residues.
Etude structurale et métabolique des acides mycoliques de Mycobacterium fortuitum
Authors: LACAVE Charlotte , LANÉELLE Marie‐Antoinette , DAFFÉ Mamadou , MONTROZIER Henri , ROLS Marie‐Pierre , ASSELINEAU Cécile
Published: March 2005

Structural and metabolic studies of the mycolic acids of Mycobacterium fortuitum. The biosynthesis of mycolic acids was studied in whole cells of Mycobacterium fortuitum. At first the structures of the main mycolates produced by the used strain were established as diunsaturated and epoxymycolates. By using [1‐14C]acetate as a radiotracer of the lipid synthesis, it was observed that the turnover of the mycolates during the exponential phase of growth of M. fortuitum is fast enough to make very difficult the identification of their precursors.

If the growth of the bacterial cells is stopped or highly diminished, by the removal of a large part of their nutritional medium, mycolate synthesis, in contrast to the synthesis of other fatty acids, is stopped as shown by incubation of the concentrated bacterial culture with [1‐14C]acetate. After removal of aliquots of the sedimented bacteria at intervals, during several hours, mycolate synthesis resumes when the cell concentration becomes lighter. In these conditions the sequence of radiolabeling of mycolates and of their potential precursors (tetracosanoate and meromycolates) can be observed. In spite of their low accumulation, tetracosanoate and meromycolates were isolated and purified and their specific radioactivity, after different incubation times, could be measured.

The results are in agreement with the hypothesis that meromycolates are condensed with tetracosanoate to produce mycolates. However, because of the large differences of isotopic dilution of these two precursors inside the mycolate molecule, this hypothesis, generally taken as evidence, has to be modified. A hypothetical pathway of the mycolate synthesis is proposed, taking into account all these observations.
Secretion of Bacillus subtilis levansucrase: a possible two‐step mechanism
Authors: PETIT‐GLATRON Marie‐Françoise , BENYAHIA Fadila , CHAMBERT Régis
Published: March 2005

The rate of exocellular levansucrase synthesis in an overproducing (sacUh) strain of Bacillus subtilis was shown to be directly proportional to the amount of two different transient forms of this enzyme located within the membrane fraction of the cells. The apparent Mr of the larger membrane form was 53000, and that of the smaller form 50000; the half‐life time of each form was estimated in vivo to be 4–6 s and 32–42 s, respectively. Ethanol treatment of the cells lead to the accumulation of the 53000‐Mr form which may represent 1.5% of total membrane proteins. This latter form, partially purified, was transformed in vitro into the 50000‐Mr form by the action of the Escherichia coli leader peptidase. These enzyme forms were quite different from the exocellular levansucrase since they showed a weak affinity for hydroxyapatite and needed complexed iron to display enzyme activity. Assuming the membrane forms were precursors of exocellular levansucrase, we propose a two‐step mechanism for the secretion process of levansucrase. The number of exoprotein synthesis/secretion sites in a B. subtilis cell is estimated to 2.5 × 104.
A DNA polymerase with unusual properties from the slime mold Physarum polycephalum
Authors: HOLLER Eggehard , FISCHER Heinrich , WEBER Cornelia , STOPPER Helga , STEGER Heinrich , SIMEK Helmut
Published: March 2005

Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE‐Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA‐agarose, blue‐Sepharose. They were separated from DNA polymerase α on phosphocellulose and from each other on heparin‐Sepharose. Form HS1 enzyme was 30–40% pure and form HS2 enzyme 60% with regard to protein contents of the preparations. Form HS2 enzyme was generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60‐kDa protein associated with either a 135‐kDa (HS1) or a 110‐kDa (HS2) DNA‐polymerizing polypeptide in a 1:1 molar stoichiometry. The biochemical function of the 60‐kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as well as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis.

The DNA polymerases resembled eucaryotic DNA polymerase β by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis.
Cytochrome c oxidase is three‐copper, two‐heme‐A protein
Authors: STEFFENS Guy C. M. , BIEWALD Rosemarie , BUSE Gerhard
Published: March 2005

Metal contents of preparations of procaryotic (Paracoccus denitrificans) and eucaryotic (beef heart) cytochome c oxidases have been determined by inductively coupled plasma atomic emission spectroscopy and shown to be stoichiometrically related to the protein contents. The results show that oxidases which possess subunits I and II have three copper atoms besides hemes a and a3 (Paracoccus denitrificans, Cu: 2.97 ± 0.08 and Fe: 2.09 ± 0.10; bovine heart, Cu: 2.83 ± 0.07 and Fe: 1.94 ± 0.12). Together with data reported for the c1aa3 oxidase from Thermus thermophilus, the following conclusions can be drawn. Subunit I binds two copper atoms and both hemes a and a3 and thus is the universal terminal oxidase of this spectral type. Subunit II binds one copper and functions as an electron conductor.

The mitochondrial respiratory complex IV binds, in addition to three copper and two hemes a, stoichiometric amounts of magnesium and zinc (bovine heart Mg: 0.98 ± 0.05 and Zn: 1.01 ± 0.04).
Chemical, immunological and biological properties of peptides like vasoactive‐intestinal‐peptide and peptide‐histidine‐isoleucinamide extracted from the venom of two lizards (Heloderma horridum and Heloderma suspectum)
Authors: VANDERMEEERS Andé , GOURLET Philippe , VANDERMEERS‐PIRET Marie‐Claire , CAUVIN Annick , DE NEEF Philippe , RATHE Jean , SVOBODA Michal , ROBBERECHT Patrock , CHRISTOPHE Jean
Published: March 2005

1. Having previously isolated helodermin, the major peptide like vasoactive‐intestinal‐peptide and peptide‐histidine‐isoleucinamidc, from the venom of the lizard Heloderma suspectum, we decided on a systematic exploration of all (VIP‐PHI)‐like peptides present in the venom of another lizard of the Helodermatidae family: Heloderma horridum.

2. Six (VIP‐PHI)‐like peptides (PHH1 to 6) were purified to homogeneity from the venom of the lizard H. horridum with PHH3 and PHH4 representing two minor forms. All peptides cross‐reacted in radio‐immunoassays for helodermin and PH1 but not for VIP. They yielded four fragments (T1 to T4) after trypsin digestion. T1, T2 and T3 showed the same retention time by reverse‐phase HPLC and the same amino acid composition; the differences were confined to T4, the C‐terminal sequence. PHH5 and PHH6 were found to be identical to synthetic helospectins I and II respectively. PHH1 and PHH3 probably resulted from a secondary modification of PHH5, while PHH2 and PHH4 derived from PHH6. Thus, the VIP‐like peptides, previously called helospectins, are in fact typical of H. horridum venom.

3. We confirmed that helodermin is the major (VIP‐PHI)‐like peptide of the venom of H.suspectum and observed its absence in H. horridum venom. Also, we found that positions 8 and 9 of helodermin are occupied by two Glu residues instead of two Gln as previously published. Helospectin‐like material was also present in H. suspectum venom but in very small amount.

4. In both venom all VIP‐like peptides were equally potent and efficient when tested for (a) their ability to occupy VIP as well as secretin receptors in rat pancreatic membranes and VIP receptors in rat liver membranes, and (b) the ensuing activation of adenylate cyclase in both membrane preparations.
Use of a polymer‐bound flavin derivative for the rapid regeneration of NAD(P)+ from NAD(P)H in dehydrogenase systems
Authors: MONTAINE France , LENDERS Jean‐Pierre , CRICHTON Robert R.
Published: March 2005

An aldehyde derivative of riboflavin was covalently attached by reductive alkylation to soluble polycationic supports. The flavopolymers so obtained were stable under operational conditions. The catalytic efficiency towards oxidation of NADH by these flavopolymers was demonstrated, and the kinetic parameters (Km and Kcat) revealed an overall catalytic efficiency (Kcat/Km) 185‐fold greater compared to riboflavin.

Various factors affecting the chemical regeneration of NAD+ from NADH such as pH, ionic strength, nature of the buffer etc. were studied. The most interesting result was the highly favourable influence of borate ions which increased the reaction rate by a factor 2–4 compared to the other buffers.

The flavopolymers are very effective for in situ recycling of NAD(P)+. With up to 300‐fold NADH → NAD+ conversions for the system using yeast alcohol dehydrogenase and up to 1500‐fold NADPH → NADP+ regenerations for the system using glucose‐6‐phosphate dehydrogenase. These flavopolymers are superior to previous chemical recycling systems.
Purification and characterization of a light‐harvesting chlorophyll‐a/b–protein of photosystem I of Lemna gibba
Authors: NECHUSHTAI Rachel , PETERSON Camille C. , PETER Gray F. , THORNBER J. Philip
Published: March 2005

The photosystem I (PSI) complex of Lemna gibba, isolated by deriphat/polyacrylamide gel electrophoresis of thylakoids solubilized in glycosidic surfactants, has been fractionated into its two chlorophyll‐protein complexes: a core component (CCI) and a light‐harvesting component (LHCI), using either non‐denaturing gel electrophoresis or ion‐exchange chromatography/sucrose gradient centrifugation. Both methods yielded an LHCI component that contained only one apoprotein of approximately 20 kDa. All the chlorophyll b and lutein of the PSI complex is associated with this LHCI preparation. The chlorophyll a/b ratio of this chlorophyll‐protein is 2.5, and lutein is essentially the only carotenoid present. While the purified LHCI from Lemna cross‐reacts with antibodies raised against spinach LHCPIb of Lam et al. [FEBS Lett. 168, 10–14 (1984)], no cross‐reactivity occurred between it and the major light‐harvesting chlorophyll‐a/b‐protein of PSII, LHCIIβ. This and a comparison of the amino acid and pigment compositions of the apoproteins of the LCHI and LCHIIβ chlorophyll‐proteins indicate that these are two distinct but similar chlorophyll‐proteins.
Regulation of the cholesterol ester cycle of cultured Leydig tumor cells
Authors: FREEMAN Dale A.
Published: March 2005

The MA‐10 Leydig tumor cells take up low‐density lipoprotein (LDL) from the medium and store the LDL‐derived cholesterol as cholesterol esters that can be subsequently mobilized and used for steroid hormone synthesis. The present studies investigate the mechanisms by which cAMP acutely regulates the cellular content of cholesterol esters. In the absence of cholesterol utilization for steroidogenesis, cAMP stimulates cholesterol ester hydrolysis and ester resynthesis proportionally. The augmentation of ester hydrolysis by cAMP is completely matched by increased activity of the acyl‐coenzyme‐A: cholesterol acyltransferase and thus does not regulate cellular cholesterol ester concentration per se. The more important action of cAMP is to interrupt the cycle of hydrolysis and ester resynthesis by decreasing cholesterol re‐esterificaton. In cells activity synthesizing steroid hormones, cholesterol reesterification is decreased by 82%. The decrease in cholesterol re‐esterification occurs because cAMP directs cholesterol normally destined for re‐esterification into steroid synthesis; simply blocking the utilization of cholesterol for steroidogenesis completely prevents net cholesterol ester hydrolysis and increases the cellular rate of cholesterol esterification.
Evidence for the transcriptional control of nitrate reductase in Candida nitratophila from in vitro translation studies
Authors: CANNONS Andrew C. , HIPKIN Charles R.
Published: March 2005

In vivo labelling and in vitro translation studies were used to study the regulation of the synthesis of nitrate reductase in the yeast Candida nitratophila. These studies showed that synthesis of the enzyme subunit took place when ammonium‐grown cells were nitrogen‐starved and this was stimulated by subsequent addition of nitrate. Ammonium‐grown cultures did not contain mRNA that could be translated into the nitrate reductase subunit in an in vitro system. Nitrate reductase mRNA could be extracted from nitrogen‐starved and nitrate cultures. Synthesis of the enzyme is apparently controlled at the level of transcription in this yeast.
Desmosine and isodesmosine as cross‐links in the hinge‐ligament protein of bivalves
Authors: KIKUCHI Yasuo , TSUCHIKURA Osamu , HIRAMA Masahiro , TAMIYA Nobuo
Published: March 2005

1. Desmosine and isodesmosine were detected in an invertebrate molluscan species, i.e. in an insoluble protein in the hinge ligament of a bivalve species, Sakhalin surf clam (Pseudocardium sachalinensis, in family Mactridae). The protein is rich in glycine and methionine S‐oxide but devoid of hydroxyproline and hydroxylysine.

2. 3,3′‐Methylenebistyrosine was also detected in the HCl hydrolysate of the hinge‐ligament protein, but it was found to be an artefact produced from tyrosine and formaldehyde derived from methionine S‐oxide during the HCl hydrolysis of the protein.
Differentiation of the drug‐binding sites of calmodulin
Authors: ZIMMER Manfred , HOFMANN Franz
Published: March 2005

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various ‘calmodulin antagonists’ for these sites was investigated. The Ki values for the inhibition of calmodulin‐activated cyclic‐nucleotide phosphodiesterase and myosin light‐chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four groups. Group I and II compounds inhibited competitively the activation of the phosphodiesterase and myosin light‐chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light‐chain kinase at identical concentrations. In contrast, group II compounds inhibited the activation of the phosphodiesterase at 5–10‐fold lower concentrations than that of myosin light‐chain kinase. Group III compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 μM and did not affect the activation of myosin light‐chain kinase.

Binding of [3H]bepridil to calmodulin under equilibrium conditions yielded one high‐affinity site (apparent Kd 0.4 μM) and four low affinity sites (apparent Kd 44 μM). Group I compounds interfered with the binding of bepridil to the high and low‐affinity sites in a competitive manner. Group II compounds interfered in a noncompetitive manner with the high‐affinity site and apparently competed only with one of the low‐affinity sites. Group III compounds did not compete with any of the bepridil‐binding sites. Nimodipine, a group III compound, bound to one site on calmodulin with a Kd value of 1.1 μM. Other dihydropyridines competed with [3H]nimodipine for this site. The group I and II compounds, trifluoperazine and prenylamine, did not affect the binding of [3H]nimodipine.

These data show that ‘calmodulin antagonists’ can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes.
Rapid purification of protein kinase C from rat brain
Authors: WOOTEN Marie W. , VANDENPLAS Michel , NEL André E.
Published: March 2005

We describe a rapid purification of protein kinase C from rat brain cytosol employing a specific substrate, protamine‐coupled to agarose. Sequential chromatography on DEAE‐Sephacel, phenyl‐Sepharose CL‐4B, and protamine‐agarose columns resulted in a 1500‐fold purification of protein kinase C. SDS‐PAGE analysis of the purified enzyme resolved a doublet protein of 77–80 kDa. This doublet was recognized by a polyclonal antiserum against protein kinase C. Proteolytic digestion of each protein band generated similar peptide fragments.

The underlying principle of the protamine sulfate purification method was also clarified. Protamine can serve as a Ca2+/phospholipid‐independent substrate. We demonstrate phosphorylation of protamine on the column: phosphorylated protamine did not bind the enzyme with the same affinity and this covalent modification was most probably responsible for releasing the bound enzyme from the column after addition of Mg2+ and ATP. The C kinase inhibitor, H7, inhibits protamine phosphorylation in a dose‐dependent fashion but does not prevent binding of the enzyme to a protamine‐agarose column. We therefore conclude that protamine interacts with the active center of the enzyme enabling it to be phosphorylated, upon which it loses its binding affinity for C kinase.
A comparative study on the phoE genes of three enterobacterial species
Authors: VAN DER LEY Peter , BEKKERS August , VAN MEERSBERGEN Joop , TOMMASSEN Jan
Published: March 2005

The cloned phoE genes from Enterobacter cloacae and Klebsiella pneumoniae are normally expressed and regulated in Escherichia coli K‐12, and their products are correctly assembled into the outer membrane. Differences between the three PhoE proteins were found with binding of two out of ten monoclonal antibodies directed against the cell‐surface‐exposed part and in pore characteristics, but not in phage receptor function. The DNA sequences of the E. cloacae and K. pneumoniae phoE genes were determined and used to predict the primary structures of the encoded proteins. In the upstream non‐coding regions, which showed more variations among the three genes than the coding regions, conserved sequences were identified which might be involved in regulation of phoE gene expression. Comparison of the predicted PhoE primary structures revealed a high degree of homology, with 81% of the amino acid residues being identical in all three proteins. Four small variable regions were found where differences are the most pronounced, corresponding to regions which were previously predicted to be exposed at the cell surface. Implications of the sequence comparison for structure‐function relationships in PhoE protein are discussed.
The calcium channel antagonists receptor from rabbit skeletal muscle
Authors: BARHANIN Jacques , COPPOLA Thierry , SCHMID Annie , BORSOTTO Marc , LAZDUNSKI Michel
Published: March 2005

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200–110, (−)[3H]D888 and d‐cis‐[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1.

Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170000 under non‐reducing conditions and of two polypeptides of Mr 140000 and 32000 under disulfide‐reducing conditions. Iodination of the protein of Mr 170000 and immunoblots experiments with antisera directed against the differents components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170000 composed of a polypeptide chain of Mr 140000 associated to one polypeptide chain of Mr 32000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29000 and 25000. Peptide mapping of these polypeptide chains and analysis of their cross‐reactivity with sera directed against the proteins of Mr 170000 and 32000 demonstrated that they were degradative products of the Mr 32000 component.

Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20–22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140‐kDa and 32‐kDa components originate from a single polypeptide chain of Mr 170000 which is cleaved by a proteolysis upon purification is discussed.
5,6‐Dichloro‐1‐β‐O‐ribofuranosylbenzimidazole induces DNA damage by interfering with DNA topoisomerase II
Authors: LÖNN Ulf , LÖNN Sigrid
Published: March 2005

We have examined DNA in cells treated with 5,6‐dichloro‐1‐β‐O‐ribofuranosylbenzimidazole (DRB), an adenosine analogue. The results show that DRB induces an partial fragmentation of DNA when the cells are lysed in dilute alkali. Fragmentation of DNA does not occur in control cells, nor in cells pretreated with novobiocin or VP‐16/VM‐26. The data show that DRB interferes with DNA topoisomerase II. In agreement with this interpretation, crude nuclear extracts of DRB‐treated cells result in reduced in vitro KCl/SDS precipitation of covalent protein‐DNA complexes. Formation of covalent complexes is typical of topoisomerase‐DNA interaction.
Overaccumulation of 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A reductase in a compactin (ML‐236B)‐resistant mouse cell line with defects in the regulation of its activity
Authors: HASUMI Keiji , YAMADA Akira , SHIMIZU Yayoi , ENDO Akira
Published: March 2005

Using a stepwise selection procedure, we have developed a line of mouse FM3A cells which is resistant to compactin (ML‐236B), a competitive inhibitor of 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme‐A (HMG‐CoA) reductase. This line, designated CR200, expressed a stable phenotype of a > 100‐fold higher resistance to the inhibitor and a 160‐fold higher HMG‐CoA reductase activity than did the parental FM3A cell line when grown in the presence of low‐density lipoprotein (LDL). In contrast to its action in the parental cells, HMG‐CoA reductase in CR200 cells was not suppressed by human LDL. The activity of both cell‐surface binding and degradation of 125I‐LDL was approximately 10% in CR200 cells as compared with that in parental cells. Affinity of the 125I‐LDL binding in CR200 cells was similar to that in the parental cells. In addition, HMG‐CoA reductase was not suppressed normally by mevalonate and 25‐hydroxycholesterol in CR200 cells; suppression was 50–60% by both compounds under conditions where suppression was about 99% in the parental cells. Sterol synthesis from [3H]mevalonate was normal in the variant cells. These observations demonstrate two defects in CR200 cells: decreased levels of LDL receptor and a defective regulation of HMG‐CoA reductase by one or more products derived from mevalonate. When grown without compactin for 21 weeks, the variant cells became slightly sensitive to compactin and concomitantly recovered partly from both defects. However, these phenotypes of CR200 cells were stable up to 15 months when exposed to compactin. These two defects may account for the overaccumulation of HMG‐CoA reductase in CR200 cells and thereby for the resistance to compactin.
Cloning and characterization of the pyrF operon of Salmonella typhimurium
Authors: THEISEN Michael , KELLN Rod A. , NEUHARD Jan
Published: March 2005

The pyrF gene of Salmonella typhimurium encoding the sixth enzyme of pyrimidine nucleotide biosynthesis, OMP decarboxylase, was isolated from a pyrF‐complementing R' factor. A 2.0‐kbp DNA fragment, generated by PvuI cleavage, was subsequently subcloned into the multicopy vector pBR322 and shown to contain the intact pyrF gene. Bacterial strains harbouring the resulting plasmid contain 15–20‐fold elevated levels of OMP decarboxylase, and these levels increase 4–5‐fold during uracil starvation.

Experiments utilizing minicells identified the gene product as a polypeptide with a molecular mass of approximately 27 kDa. Furthermore, it was found that the pyrF gene is expressed as the first gene of a bicistronic operon, wherein the second gene encodes an 11‐kDa polypeptide of unknown functions. The complete nucleotide sequence of the pyrF operon was determined. An open reading frame, encoding a polypeptide with a calculated molecular mass of 26213 Da, was deduced to be the coding region for pyrF. Another open reading frame, with a translational start codon which overlaps the translational stop codons of the pyrF gene, encodes a polypeptide of 11513 Da. This open reading frame represents the coding region for the second gene of the operon, orfF. S1‐nuclease mapping indicated that pyrF transcription is initiated 54 bases upstream of the translational start. The leader region does not show any features resembling the attenuators found preceding the pyrBI operon and the pyrE gene.
Biological role of the pneumococcal amidase
Authors: RONDA Concepción , GARCÍA José Luis , GARCÍA Ernesto , SÁNCHEZ‐PUELLES José M. , LÓPEZ Rubens
Published: March 2005

A pneumococcal recombinant plasmid, pRG2, containing the lytA gene that codes for the pneumococcal N‐acetylmuramoyl‐L‐alanine amidase has been constructed using the pneumococcal plasmid pLS1 as a vector. pRG2 was introduced by genetic transformation into a mutant of Streptococcus pneumoniae (M31) that has a complete deletion of the lytA gene. The transformed strain (M51) grew at a normal growth rate as ‘diplo’ cells and underwent autolysis at the end of the exponential phase of growth, two properties that had been lost in the deleted mutant M31. M51 lysed very rapidly at the end of the exponential phase when the cells were grown in choline‐containing medium probably because of the higher level of amidase activity present in this strain as compared to the lysis‐prone strain M11. These findings show that the expression of the plasmid‐linked gene was placed under the mechanism(s) of control of the cell during the exponential phase. Our results demonstrate that the physiological role of the pneumococcal amidase was to catalyze the separation of the daughter cells at the end of the cell division to produce diplo cells; in addition we have also confimed the basic role of this autolysin in the bacteriolytic nature of β‐lactam antibiotics.