bluuebunny/crossref_metadata_2025 huggingface dataset:
Limited Proteolysis of Porine‐Muscle Latic Dehydrogenase by Thermolysin during Reconstitution Yields Dimers
Authors: GIRG Rainer , RUDOLPH Rainer , JAENICKE Rainer
Published: March 2005

Reassociation of lactic dehydrogenase from porcine skeletal muscle at 10°C was studied by limited proteolysis during reconstitution, applying thermolysin as proteolytic enzyme. Proteolysis was achieved by short incubation of the reassociating enzyme with the protease in a ratio lactic dehydrogenase: thermolysin = 10:1 (0.1 M sodium phosphate buffer pH 7.6 plus 1mM dithioerythritol).

Thermolysin was found to satisfy all the oligatory requirements for proteolytic studies of reconstituting enzymes:(a) the protease is essentially inactive towards native lactic dehydrogenase;(b) it splits intermediates of reconstitution at a high rate; (c) it can be inactivated instantaneously y the addition of 10 mM EDTA.

As demonstrated by gel chromatography, thermolysin digestion stailizes dimers of porcine muscle lactic dehydrogenase as intermediates of reconstitution. These ‘dimers’ consist of virtually intact polypeptide chains of molecular weight about 35000, apart from nicked subunits with molecular weights of 18000 and 12000, as determined y sodium dodecyl sulfate/polyacrylamide gel electrophoresis.

Anallyzing the kinetics of reassociation by thermolysin digestion at various times during reconstitution, and susequent gel filtration, the dimer → tetramer transition is found to be rate‐limiting for both reassociation and reactivation. This result corroborates a previously proposed model for the reconstitution of lactic dehydrogenase, based on either cross‐linking experiments [R. Hermann et al. (1979) Nature (Lond.) 277, 243‐245], or the reassembly of dimeric intermediates of dissociation [R. Jaenike et al. Eur. J. Biochem. 114, 525–531].
Expression of mRNA Coding for Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: MacLEOD Alexander R.
Published: March 2005

The expression of the mRNA coding for glyceraldehydes‐3‐phosphate dehydrogenase has been studied during embryonic development of the chicken.In each tissue examined, only one mRna species coding for a single glyceraldehydes‐3‐phosphate dehydrogenase subunt could be detected using translation in vitro with RNA plotting and hybridization. The mRna species coding for glyceraldehydes‐3phosphate dehydrogenase subunits in different chicken tissues have identical electrophoretic mobilities suggesting that they are structurally vey similar if not identical.
Stereopecificity and Other Properties of a Novel Secondary‐Alcohol‐Specific Alcohol Dehydrogenase
Authors: HOU Ching T. , PATEL Ramesh , BARNABE Nancy , MARCZAK Irene
Published: March 2005

NAD‐ dependent alcohol dehydrogenase from the methanol‐grown Methylcoccus sp. CRL MI (type I membrane). Methylosinus trichosporium OB3b (type II membrane), Methylobacterium organophillum CRL 26 (type II membrane, facultative methylotroph) Pseudomonas sp. ATCC 21439, and Pichia pastoris Y‐55 are secondary alcohol‐specific and that from P pastoris Y‐7556 is not. This novel secondary‐alcohol‐specific alcohol dehydrogenase (secondary‐alcohol dehydrogenase) has been purified from methanol‐grown Pseudomonas sp. ATCC 21439. Secondary‐alcohol dehydrogenase shows a single protein band on acrylamide gel electrophoresis and has a molecular weigth of 95000. It consists of two subunits of Mr 48000 daltons and two atoms of zinc per molecule of enzyme protein. It oxidizes secondary alcohols, notably 2‐propanol and 2‐butanol. Primary alcohols are not oxidized. The pH and temperature optima for secondary‐alcohol dehydrogenase are 8‐9, and 30‐35 °C, respectively. The activation enrgy calculated is 82.8kJ.Secondary‐alcohol dehydrogenase also catalyzes the reduction of methyl ketones to their corresponding 2‐alcohols in the presence of NADH (a reverse reaction). The Km values at 25 °C in the forward reaction for 2‐butanol, (2R)‐(−)‐butan‐2‐ol, and NAD, and in the reverse reaction for 2‐butanone and NADH are 2.5×10−4 M, 1.1×10−5M, 1.98×10−4M, and 2.1×10−6M, respectively. The secondary‐alcohol dehydrogenase activity was inhibited by metal‐chelating agents and by strong thio regents such as p‐hydroxymercuribenzoate and 5,5′‐dithiobis(2‐nitrobenzoic acid).The substrate specificity, and mobility on gel electrophoresis of secondary‐alcohol dehydrogenase oxidizes preferentially the (−)‐2‐butanol. This different from primary‐aclcohol dehydrogenase from bakers' yeast which oxidizes only the(+)‐2‐butanol. This may be explained in terms of the structure of the enzymes.
A Role in vivo for Penicillin‐Binding Protein‐4 of Staphylococcus aureus
Authors: WYKE Anne W. , WARD J.Barrie , HAYES Michael V. , CURTIS Nigel A.C.
Published: March 2005

The degree of cross‐linking of the peotidoglycan of Staphylococcus aureus H and mutants lacking penicillin‐binding proteins 1 and 4 was studies. No major changes were ovserved in organisms lacking protein 1 whereas oloss of protein 4 was accompanied by a marked reduction in the degree of cross‐linked and the absence of a membrane‐bound ‘model’ transpeptidase activity. A smimlar effect was achieved when cultures of the staphylococci were treated with the β‐lactam antibiotic cefoxitin. At low concentrations (0.05μg ml‐1) cefoxition shows highest affinity for protein 4 to which it appears to bind irreversibly. Treatment of the mutant lacking protein 4 with this concentration of the antibiotic did not affect the degree of cross‐linkage.

The possibility that the decrease in cross‐linkage was a consequence of DD‐carboxypeptidase activity on peptidoglycan precursors was investigated. Although both S. aureus H and the mutants possessed such activity it was insensitive to benzylpenicillin and cefoxition and the role of this enzyme(s) in peptidoglycan biosynthesis remains unknown.

We conclude that in vivo protein 4 acts as a transpeptidase involved in the secondary cross‐linking of peptidoglycan and this activity is necessary to achieve the high degree of cross‐linkage observed in the peptidoglycan of staphylococci.
The Extent of Histone Acetylation Induced by Butyrate and the Turnover of Acetyl Groups Depends on the Nature of the Cell Line
Authors: SCHRÖTER Hennrik , GÓMEZ‐LIRA M. Macarena , PLANK Karin‐Heide , BODE Jügern
Published: March 2005

Cells possessing widely different physiological and morphological features have been treated with substances known to stimulate the differentiation of erythroleukemia cells. Only short fatty acids are capable of causing a hyperacetylation of the core histones and of enhancing the level of an H1‐like protein in Chinese hamster ovary cells. While the time courses of a butyrate‐mediated acetylation are similar for all cells, the maximum histone acetyl contents are much higher for the transformed cell of a given type. A withdrawal of butyrate rapidly (within 45 min) gives rise to a ‘hypoacetylated state’ for fibroblasts and transformed fibroblast (epithelial) cells from which there is a slow recovery Lymphoid cells, on the other hand, display a marked persistence of the highly aaacetylated forms of histone H4.
Cytochromes P‐450 LM3b and LM4 in Biosynthesis of Bile Acids
Authors: BOSTRÖM Hans , HANSSON Ronnie , JÖNSSON Karl‐Henrik , WIKVALL Kjell
Published: March 2005

Cytochromes P‐450 LM3b and LM4 were prepared from untreated and cholestyramine‐treated rabbits. The catalytic properties of these cytochrome P‐450 fractions towards substrates in bile acid biosynthesis were studied reconstituted systems containing NADPH–cytochrome P‐450 reductase and phospholipid. Cytochrome P‐450 LM3b showed no hydroxylase activity towards cholesterol and only low activity towards some other C27‐steroids whereas it catalyzed efficient hydroxylation of testosterone and demethylation of ethylmorphine. Preparations of cytochrome P‐450 LM4 catalyzed hydroxylation of cholesterol and other C27‐steroids more efficiently than microsomes. Cytochrome b5 had no stimulatory effect on the C27‐steroid hydroxylase activities.
The Conservation of DNA Sequences over Very Long Periods of Evolutionary Time
Authors: FILER David , DHAR Ravi , FURANO Anthony V.
Published: March 2005

In the present study we tried to determine whether the presence of DNA sequences homologous to the Escherichia coli tuf gene (encodes peptide chain elongation factor Tu) in many taxonomically‐unrelated prokaryotes is due to selective pressure for these sequences or due to the transfer of chromosomal material subsequent to the divergence of the genera from their progenitors. We found that the degree of sequence homology to the DNA immediately adjacent to the E. coli tuf A gene is either nonexistent or much less than that found for the tuf gene. Furthermore, the tuf‐homologous sequences present in one prokaryote were found to be in large part the same as or a subset of those present in others. That is, various prokaryotes share a common subset of tuf‐homologous sequences. These findings suggest that strong selective pressure and not recent intergeneric chromosomal transfer is responsible for the ubiquitous presence of certain tuf‐homologous sequences. Because the genetic code is degenerate, DNA sequence need not be conserved to conserve protein sequence. Therefore, if the only function of these sequences is to encode protein, their persistence must mean that in some instances codon sequence is selected for.
The Protochlorophyllide Holochrome of Barley (Hordeum Vulgare L.)
Authors: APEL Klaus
Published: March 2005

During the illumination of dark‐grown barley plants light induces a rapid decrease of a translatable mRNA which codes for a polypeptide of Mr44000. This component was identified as a precursor of the NADPH: protochlorophyllide oxidoreductase. The precursor has an Mrlarger than the authentic protein by approximately 8000.

The light‐induced change in the level of translatable mRNA can be induced by a 15‐s red‐light pulse followed by 5 h of darkness. The red‐light effect is reversed by a subsequent far‐red‐light treatment. It is concluded that the light‐induced decline of translatable mRNA for the NADPH: protochlorophyllide oxidoreductase is controlled by phytochrome. The significance of this finding for present concepts of light‐dependent control of chloroplast development and chlorophyll synthesis is discussed.
Induction of Polymerization of Purified Tubulin by Sulfonate Buffers
Authors: WAXMAN Phyllis G. , DEL CAMPO Anthony A. , LOWE Michael C. , HAMEL Ernest
Published: March 2005

Interactions of both purified tubulin and microtubule protein (tubulin plus associated proteins) with two commonly used sulfonate buffers were examined. 1,4‐Piperazineethanesulfonate(Pipes) and 4‐morpholineethane‐sulfonate (Mes) at high concentrations induce the polymerization of purified tubulin in reactions requiring only buffer, tubulin and GTP. While both reactions were temperature‐dependent, cold‐reversible and inhibited by GDP, colchicine or Ca2+, there were significant differences between them. Substantially lower tubulin and buffer concentrations were required for Pipes‐induced polymerization; and turbidity was much more intense in the Pipes‐induced than in the Mes‐induced reaction at the same protein concentration. Electron microscopy demonstrated that for the most part typical smooth‐walled microtubules were formed in Mes, while aberrant forms were the predominant structures formed in Pipes.

When the polymerization of microtubule protein was examined as a function of buffer concentration, biphasic patterns were observed with both Pipes and Mes: polymerization occurred at both and high, but not intermediate, buffer concentrations. The turbidity observed at high concentrations of Pipes greatly exceeded that at low concentrations. With Mes, equivalent turbidity developed at both high and low buffer concentrations. Although associated proteins copolymerized with tubulin at low buffer concentrations, they were excluded from the polymerized material at high buffer concentrations.

Pipes and Mes were compared to sodium phosphate, Tris/HCl and imidazole/HCl buffers at 0.1 M is several polymerization systems using both purified tubulin and microtubule protein. The sulfonate buffers were invariably associated with more vigorous reactions than the other buffers.
On the Rolw of the Carbohydrate Side Chains of Human Plasminogen in Its Interaction with α2‐Antiplasmin and Fibrin
Authors: LIJNEN H. Roger , VAN HOEF Berthe , COLLEN Désiré
Published: March 2005

The Dissociation constants (Kd) for the interaction between different plasminogen derivatives and α2‐antiplasmin were determined as the concentrations of ligand that decrease the rate of the reaction between plamin and α2‐antiplasminby 50%. The Kd of Lys‐plasminogen (residues 77–790) type I (with a glucosamine‐based oligosaccharide chain attached to Asn‐288) and type II (with an unsubstituted Asn‐288) was found to be 1.9 μM and 0.2 μM respectively. The isolated plasminogen type I fragments containg the high‐affinity lysine‐binding site (representing residues 79–337 with glucosamine‐based carbohydrate on Asn‐288 or residues 79–353 with glucosamine‐based carbohydrate on Asn‐288 and galactosamine‐based carbohydrate on Thr‐345) both had a dissociation constant of 1.3 μM. The isolated plasminogen type II fragments containg the high‐affinity lysine‐binding site (residues 79–337 without carbohydrate or residues 79–353 with galactosamine‐based carbohydrate on Thr‐345) both had a dissociation constant of 0.24μM. These findings indicate that the difference in Kd is related to the presence of an oligosaccharide side chain on Asn‐288 but is unrelated to the presence of carbohydrate on Thr‐345. The dissociation constants of type I and type II Glu‐plasminogen (residues 1–790) were 4.9 μM and 2.9 μM. These higher values and smaller differences between type I and type II can probably be explained by the presence of the NH2‐terminal preactivation peptide (residues 1–76) which interacts with the high‐affinity lysine‐binding site in Glu‐plasminogen.

Using 125I‐labelled plasminogen derivatives, direct binding studies to fibrin were performed. This revealed that the binding of the purified fragments obtained from type II plasminogen was 7‐times higher than that of fragments obtained from type I plasminogen. For both Glu‐plasminogen and Lys‐plasminogen no difference in extent of binding was observed for type I and type II.

These finding indicate that the presence of a carbohydrate chain on Asn‐288 to both α2‐antiplasmin and fibrin. This decrease was still observed for the interaction between Lys‐plasminogen and α2‐antiplasmin, but not for the extent of binding to fibrin, suggesting that other interactions than via the high‐affinity lysine‐binding site might contribute in the binding of plasminogen to fibrin.
Tryptic Modification of Human Serum Low‐Density Lipoprotein Leads to Altered Rates of Turnover and Intravascular Transformation in the Guinea Pig
Authors: WEECH Philip K. , CHAPMAN M. John , MILLS Gervase L. , GOLDSTEIN Sonia
Published: March 2005

The metabolism of human serum low‐density lipoprotein (LDL) and its trypsin‐treated counterpart have been compared in the guinea pig in vivo. Removal of surface‐exposed protein from the lipoprotein particle in this way resulted in significant modification of its metabolism in guinea pigs in vivo. Limited trypsinisation of LDL permitted removal of 20–25% of its protein moiety; trypsinised LDL was deficient in lysine and arginine residues (25–30% of each removed). The modified particle retained its basic structural features, such as internal molecular architecture, but displayed an elevated net negative surface charge and diminished immunological reactivity. Following intravascular injection of iodinated LDL (131I) and trypsinised LDL (125I) into the same animal, the two lipoproteins displayed biexponential decays; the rate constants for the plasma turnover of LDL and trypsinised LDL were significantly different (P < 0.05), trypsinished LDL exhibiting a slower disappearance from the circulation. Density‐gradient ultracentrifugation revealed marked elevation in the modal densities of both LDL and trypsinised LDL upon metabolism in vivo, although the rate of increase was greater for trypsinised LDL than LDL in each case (average increment 0.022 g/ml and 0.014 g/ml at 24 h respectively). The diminished plasma clearance of trysinised LDL as compared to the native human and guinea pig LDL indicated that sites required for the cellular recognition and uptake of the LDL particle reside in its surface‐exposed, trypsin‐accessible protein. Furthermore, such protein appears to play a central role in regulating the intravascular processes by which the lipid content of LDL is diminished, and by which it is transformed to a particle of higher density.
Effects of Tunicamycin on the Cell‐Surface Binding, Internalization and Degradation of Low‐Density Lipoproteins in Human Fibroblasts
Authors: CHATTERJEE Subroto , SEKERKE Catherine S. , KWITEROVICH Peter O.
Published: March 2005

The effect of tunicamycin, an inhibitor of protein glycosylation via the dolichol pathway, on the binding, internalization and degradation of low‐density lippoproteins was studied in cultured human fibroblasts. Glycoprotein synthesis was inhibited 50% in tunicamycin‐treated cells, while protein and nucleic acid synthesis were minimally inhibited. The binding of 125I‐labeled low‐ density lipoproteins (measured as dextran‐sulfate‐releasable 125I‐lipoproteins) in the tunicamycin‐treated cells at 4°C was saturable, but the amount bound was only one‐half that found in control cells. When these cells were warmed to 37°C for 30 min, the amounts of lipoproteins that were internalized (dextran‐sulfate‐resistant 125I‐labeled low‐density lipoproteins) and degraded wre proportional to the amounts bound in both the tunicamycin‐treated and control cells. Tunicamycin also had no effect on the rate of internaliztion of 125I‐labeled low‐density lipoproteins that were displaced by increasing concentrations of unlabeled low‐density lipoproteins were the same in both tunicamycin and control cells, an effect that was accompanied by similar patterns of decrease in the internalization and degradation of 125I‐labeled low density lipoproteins; high‐density lipoproteins and fettuin had no inhibitory effect on these processes. Tunicamycin did not inhibit the degradation of 125I‐labeled low‐density lipoproteins in homogenates of fibroblasts at pH 4.0. The data suggest that tunicamycin affects the number rather than the functional capacity of receptors on the cell surfacce since the kinetics of binding of 125I‐labeled low‐density lipoproteins to tunicamycin‐treated cells is normal and the bound lipoproteins are both internalized and degraded normally. The data further suggest a role of the dolichol‐saccharide pathway in the normal intracellular metabolism of the receptor for low‐density lipoproteins.
1H Nuclear‐Magnetic‐Resonance Studies of the Conformation of Cardiotoxin VII2 from Naja mossambica mossambica
Authors: STEINMETZ Wayne E. , MOONEN Chrit , KUMAR Anil , LAZDUNSKI Michel , VISSER Leon , CARLSSON Fritz H. H. , WÜTHRICH Kurt
Published: March 2005

The membrane toxin VII 2 from the venom of Naja mossambica mossambica was investigated in aqueous solution by one‐dimensional and two‐dimensional high‐resolution nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization included identification of the complete spin systems for several amino acid residues, nuclear Overhauser effect measurements, the use of chemically induced dynamic nuclear polarization and studies of the pH dependence of the NMR spectrum. Data from homologous toxins, in particular direct lytic factor 12B from Haemachatus haemachatus, were used to establish assignments of aromatic and methyl proton resonances. From these experiments a short, triple‐stranded fragment of antiparalles β structure could be determined, which includes the residues 23–27, 43–46 and 60–62. Furthermore, the nuclear Overhauser effect measurements indicate close proximity in the protein conformation of the aromatic rings of Trp‐14, Tyr‐25 and Tyr‐59, and the side chain of Ile‐46.
Origin and Characterization of Short DNA Chains in Escherichia coli
Authors: SIEGMANN Deborah W. , WERNER Rudolf
Published: March 2005

One of the major problems in the study of DNA replication involves the presence of numerous short, non‐nascent DNA chains in the cell. Such chains often contaminate nascent DNA preparations, making accurate analysis of the replicative DNA difficult. This complication can be avoided by the use of hydroxyapatite chromatography. Previous results from this laboratory had shown that short nascent DNA preparation which containsonly nascent DNA and is essentially free of non‐nascent chains. 5′‐Terminal labeling patterns and molecular weight distributions show that only nascent DNA chains form the DNA‐protein complex and can therefore be separated from non‐nasent chains if the isolation is performed under non‐denaturing conditions. The protein involved in the complex appears to be rather specific for DNA chains in the replication fork since short chains resulting from the excision of dUMP immediately after replication also form the DNA‐protein complex.

Using this isolation technique, 5′‐terminal nucleotides of nascent DNA was pulse–labeled in several ways, purified by hydroxyapatite chromatography, and labeled at the 5′ end with [32] phosphate. Two‐dimensional thin‐layer chromatography of the 32P‐labeled nucleotides, obtained after enzymatic hydrolysis, revealed that all four deoxyribonucleotides are present at the 5′ end in approximately equal amounts. In most cases, this 5′‐terminal nucleotide distribution does not vary significantly, even when the conditions of pulse‐labeling are changed. Only if the cells are allowed to run out of thymine is a variation in the relative amounts of 5′‐end nucleotides. In this case, the amount of dTMP at the 5′ end decreases signigicantly, refleting the low thymine levels and indicating that the cell can compensate for this deficiency by utilizing other available nucleotides. From these results, it appears that DNA chain initiation is essentially random with respect to the nucleotide used in the initiation process.
Synthesis in vitro and Translocation of Apolipoprotein AI across Microsomal Vesicles
Authors: STOFFEL Wilhelm , BLOBEL Günter , WALTER Peter
Published: March 2005

Apolipoprotein AI of rat has been synthesized in a cell‐free wheat germ system and contranslationally translocated into dog pancreas microsomal vesicles. Translocation is accompanied by cleavage of a signal sequence of 18 amino acid residues and is dependent on the recently purified signal recognition protein.
Phosphorylation of Ribosomal Protein S6
Authors: NIELSEN Peter J. , DUNCAN Roger , McCONKEY Edwin H.
Published: March 2005

The time course of S6 phosphorylation and several aspects of protein synthesis have been studied in suspension cultures of HeLa cells, following transfer to fresh medium and serum. The phosphorylation of S6 is not temporally correlated with changes in polypeptide initiation and elongation rates, as judged from polysome profiles. Phosphorylation of S6 can be maximal within 30 min after transfer; elongation and initiation rates increase coordinately and more slowly, becoming maximal about 6 h after transfer, a time at which the net phosphorylation of protein S6 is greatly reduced or negligible. Recruitment of messenger RNA into polysomes is another response to fresh medium and serum; this response occurs almost as rapidly as the phosphorylation of S6. We suggest that the phosphorylation of S6 may play a role in messenger RNA recruitment.
Purification and Properties of an Endonuclease from the Mitochondrion of Saccharomyces cerevisiae
Authors: ROSAMOND John
Published: March 2005

An endonuclease, which is found only in the mitochondrion of the yeast Saccharomyces cerevisiae, has been purified. The protein has a sedimentation coefficient of 6.3 S, equivalent to a molecular weight of 105000. The enzyme is active at pH 7.6, when it degrades single‐stranded DNA about 10‐times faster than double‐stranded DNA, but at pH 5.4 only double‐stranded DNA is degraded. In both cases the enzyme acts endonucleolytically, breaking a single phosphodiester bond at a random location within the DNA substrate. Mn2+ or Mg2+ are required for activity; Ca2+ and Zn2+ are ineffective cofactors. Enzyme activity at pH 7.6 is severely inhibited by low concentrations of NaCl or KCl, while activity at pH 5.4 is unaffected by salt. Ethidium bromide inhibits both the DNase activity at pH 5.4 and the activity with single‐stranded DNA at pH 7.6, but has no effect on the DNase activity with double‐stranded DNA at pH 7.6.
The Effect of Inhibition of (ADP‐ribose)n Biosynthesis on DNA Repair Assayed by the Nucleoid Technique
Authors: DURKACZ Barbara W. , IRWIN Judy , SHALL Sidney
Published: March 2005

DNA damage and repair was assayed by the loss and restoration of DNA supercoiling in nucleoids. This technique was used to assess the effects of inhibition of (ADP‐ribose)n biosynthesis by 3‐aminobenzamide on the capacity to repair DNA of mouse leukaemia L1210 cells following damage by γ‐irradiation and by the mono‐ functional alkylating agent, dimethyl sulphate. 3‐Aminobenzamide partially inhibits restoration of supercoiling following γ‐irradiation and dimethyl sulphate treatment, but inhibits neither the enzymic incision events leading to breaks in the DNA nor the repair synthesis. This inhibition of repair can be observed at very low doses of damaging agents. These observations confirm and extend the evidence that (ADP‐ribose)n biosynthesis is required for efficient cellular recovery from DNA damage. In particular, the nucleoid technique permits the demonstration that 3‐aminobenzamide inhibits DNA repair after γ‐radiation; it is not possible to draw this unequivocal conclusion with the data from alkaline sucrose gradients because this technique is too insensitive.

1‐β‐D‐Arabinofuranosylcytosine, which inhibits seiniconservative DNA replication, also retards repair, and this effect can be reversed by the addition of deoxycytidine. The inhibitors of DNA excision repair, arabinosyl‐ cytosine, hydroxyurea or 3‐aminobenzoamide increase the steady‐state number of DNA breaks. Thus, they can be used to enhance even further the sensitivity of the nucleoid assay of repair.
Studies on the Role of a Nucleoside‐Phosphate‐Binding Site of Diphtheria Toxin in the Binding of Toxin to Vero Cells or Liposomes
Authors: BOQUET Patrice , DUFLOT Edith
Published: March 2005

Using Fab antibody fragments directed against protein crm 45 (non‐toxic cross‐reacting material serologi‐cally‐related to diphtheria toxin mutant protein, the product of a non‐sens mutation in the diphtheria toxin gene) or the B 45 domain of this molecule (the remaining portion of the toxin B fragment present in protein (crm 45), we have shown that the diphtheria toxin polyphosphate‐binding site (P site) [Lory and Collier (1980) Proc. Nut1 Acad. Sci. USA, 1, 267–271], evidenced by the ability of the toxin to bind ATP, is affected upon incubation of this protein with the Fab fragment of anti‐protein (crm 45) IgG but not with the Fab fragment of anti‐polypeptide (B 45) IgG. After incubation with an excess of the Fab fragment of anti‐polypeptide (B 45) IgG, 125I‐labeled toxin does not bind to toxin‐sensitive Vero cells. We therefore suggest that the P site is not by itself the binding domain of diphtheria toxin to the cell membrane receptor and that the region of the toxin molecule devoted to this particular activity overlapped probably on the B 45 polypeptide. In contrast with the results obtained by Alving et al. (Proc. Nat1 Acad. Sci. USA, 1980, 4, 1986–1990) with or without a functional P site, diphtheria toxin is able to bind liposomes. However interaction with phospholipid vesicles with toxin is blocked upon incubation of the protein with the Fab fragment of anti‐polypeptide (B 45) IgG. Our data indicate that the P site of toxin does not participate in the ability of this molecule to bind liposomes.
Transformations of Zearalenone and α‐Zearalanol by Homogenates of Human Prostate Glands
Authors: THOUVENOT Daniel , MORFIN Robert , DI STEFANO Sylvie , PICART Daniel
Published: March 2005

The present study applied high‐pressure liquid chromatography, gas chromatography/mass spectrometry and crystallizations to constant specific activity for identification of the radiometabolites of tritiated zearalenone and a‐zearalanol in homogenates of human prostate glands. Radiolabelled zearalenone or α‐zearalanol (6.3 μM) was incubated for 30 min at 37°C with prostatic homogenate in 5 ml 0.067 M phosphate buffer (pH 7.4) containing 0.5 mM oxidized or reduced NAD or NADP. Reduction of zearalenone occurred only with NADPH while oxidation of α‐zearalanol was favoured both by NADP+ and NAD+. Separation of the substrates from their metabolites by high‐pressure liquid chromatography showed the radioactivity of the metabolite of zearalcnone to be associated with the α‐zearalenol carrier while that of the α‐zearalanol metabolite was found with the zearalanone carrier. Crystallizations to constant specific activity after isotopic dilution and gas chromatography mass spectrometry confirmed the identity and the radiochemical purity of the a‐zearalanol substrate and of its zearalanone metabolite. In contrast, the poor radiochemical purity of the zearalenone substrate and of its α‐zearalenol metabolite was explained by the presence of large quantities of cis‐isomer contaminants which were identified by gas chromatography/mass spectrometry. Formation of the cis‐isomer of zearalenone was shown to occur spontaneously in incubations with boiled homogenates. The other stereospecific reductions or oxidations of the substrates were due to the prostatic enzymes which could be those involved in the Clg‐steroid hormone metabolism.
The Cell‐Free Protein Synthesis System from the ‘Slime’ Mutant of Neurospora crassa
Authors: SZCZIŃ‐SKORUPA Elzȩbieta , FILIPOWICZ Witold , PASZEWSKI Andrzej
Published: March 2005

A simple procedure for preparation of a cell‐free protein synthesis system (23000 × g supernatant) from protoplast‐like ‘slime’ mutant of Neurospora crassa is described. A variety of messenger RNAs of viral and cellular origin could be efficiently and faithfully translated in this system into proteins with M, as large as 180000.

The importance of the 7‐methylguanosine cap for mRNA translation in the Neurospora system was studied in detail using the cap analogs and chemically decapped messengers. As in the case of reticulocyte lysate or wheat germ extract, the extent of m7G requirement for mRNA translation in a fungal extract strongly depended on translation conditions such as incubation temperature or concentration of potassium ions, mRNA and 23000 × g supernatant protein.
Effects of Cations on High‐Affinity and Low‐ Affinity ATP‐Binding Sites of (Na+, K+)‐ATPase as S+tudied by Disulfides of Thioinosine Triphosphate and Its Analogue
Authors: PATZELT‐WENCZLER Rosemaric , MERTENS Wolfgang
Published: March 2005

Binding of the disulfides of thioinosine triphosphate and thioinosine 5′‐[β,γ‐methylene]triphosphate, (SnoPPP), and (SnoPP[CH2]P)2, to the (Na+,K+)‐ATPase in the presence of 10mM EDTA results in an inactivation of the enzyme.

K+, Rb+ and Cs+ protect the high‐affinity and low‐affinity binding sites of (Na+,K+)‐ATPase against inactivation by (SnoPPP), and (SnoPP[CH2]P)2. The protection constants increase with the atomic radii of the monovalent cations.

The protective effects of low concentrations of K +, Rb+ and Cs+ on the inactivation of the (Na+,K+)‐ATPase by (SnoPP[CH2]P)2 are smaller than the effects on the inactivation by (SnoPPP)2. The electronegativity of the bridge atom between β and θ phosphate groups obviously has a function on the mode of action of monovalent cations.

High concentrations of K+, Rb+ and Cs+ accelerate the inactivation of the high‐affinity and low‐affinity binding sites of the (Na+,K+)‐ATPase by (SnoPP[CH2]P)2.

Na+ concentrations below 10 mM advance the inactivation of the high‐affinity and low‐affinity binding sites of the (Na+, Kplus;)‐ATPase by (SnoPPP)2 and (SnoPP[CH2]P)2. Na+ concentrations above 10 mM protect against inactivation.

K+ raises the activatingeffect in the presence of low concentrations of Na+ by inactivation through (SnoPPP)2 and (SnoPP[CH2]P)2. The protective effect of higher concentrations of Na+ is amplified by K+, when using (SnoPPP)2, whereas it is reduced in the case of (SnoPP[CH2]P)2.

Mg2+ concentrations up to 5 mM advance the inactivation of the high‐affinity and low‐affinity binding sites of the (Na+, K+)‐ATPase by (SnoPPP)2 and (SnoPP[CH2]P)2. Higher concentrations of Mg2+ have a protective effect.

The monovalent cations (Na+, K+, Rb+, Cs+) and Mg2+ act in a noncompetitive manner with respect to nucleotide binding. It is therefore concluded, that they are effective by inducing conformational changes of the enzyme.
Effects of Borohydride‐Treated Oligomycins on Processes of Energy Transduction in Mitochondria
Authors: RAMIREZ Fausto , MARECEK James F. , KANTOR Thomas V. , OKAZAKI Hiroshi , TU Shu‐I
Published: March 2005

Sodium borohydride in ethanol solution under mild conditions brings about the stepwise reduction of the 7‐keto and the 11‐keto groups of rutamycin and the oligomycins to the corresponding hydroxyl groups without further alterations of the macrocyclic lactone structure or other features of the molecule. The reduced compounds, as well as the parent antibiotics, inhibit the ADP‐dependent (state 3) respiration, and the Pi formation and proton extrusion that are linked to ATP hydrolysis, but have no effect on other respiration‐linked activities in intact rat liver mitochondria. Analogous inhibitory effects of borohydride‐treated antibiotics are also observed in rat‐liver submitochondrial particles. The reduced compounds are less potent inhibitors than the parent antibiotics. The reduced compounds are more efficient as inhibitors of Pi formation stimulated by conventional uncouplers (e.g. 2,4‐dinitrophenol), than of Pi formation stimulated by certain amine‐fluorescamine modifiers (e.g.) the benzylamine‐fluorescamine compound. In contrast, the parent antibiotics are unable to discriminate between uncoupler‐stimulated and modifier‐stimulated Pi formation. It is suggested that rutamycin and the oligomycins bind to H+‐ATPase as a result of hydrogen bonding to, at least, the 7‐keto and/or the 11‐keto groups of the antibiotics. When these keto groups are reduced to hydroxyl groups the hydrogen‐bonding is less efficient due to the pronounced directional characteristic of hydrogen‐bonding to keto groups.
Control of Ketogenesis and Fatty‐Acid Synthesis at the Mitochondrial Branch‐Point for Acetyl‐CoA in the Chick Liver Cell: Effect of Adenosine 3′,5′‐Monophosphate
Authors: MOONEY Robert A. , LANE M. Daniel
Published: March 2005

Previous work has shown that dibutyryl adenosine 3′,5′‐monophosphate (Bt2cAMP) markedly activates ketogenesis from fatty acids released during the mobilization of cytoplasmic triacylglycerol‐rich vesicles in the chick liver cell. In the present investigation, activation of the formation of [14C]acetoacetate and 3‐[14C]hydroxybutyrate from [1‐14C]oleate by the cyclic nucleotide was shown to occur at two mitochondrial sites: (a) the l‐(–)‐carnitine‐dependent translocation of the fatty acid across the inner mitochondrial membrane and (b) the intramitochondrial metabolism of acetyl‐CoA.

The hypothesis was tested that cAMP regulates ketogenesis and fatty acid synthesis by controlling acetyl‐CoA flux at the mitochondrial branch‐point leading into the 3‐hydroxy‐3‐methylglutaryl‐CoA cycle or to citrate formation. Bt2cAMP caused an immediate activation of acetoacetate formation and a concomitant inhibition of fatty acid synthesis from acetate. Evidence is presented that these effects are due to a cyclic‐nucleotide‐induced fall in the mitochondrial concentration of oxaloacetate. An assay for oxaloacetate, sufficiently sensitive to analyze extracts of cells in monolayer culture, was employed. With this assay, Bt, cAMP was shown to cause a > 90% decrease in cellular oxaloacetate concentration. The calculated level to which intramitochondrial oxaloacetate fell was below the Km for oxaloacetate in the citrate‐synthase‐catalyzed reaction and, therefore, was sufficient to curtail citrate formation.

Lactate, a precursor of oxaloacetate and an antiketogenic agent, reversed the effects of Bt2cAMP. Under conditions where glucose was sole precursor of oxaloacetate and citrate, the activation of ketogenesis and the inhibition of fatty acid synthesis by Bt2cAMP were reversed by lactate. Lactate also reversed the Bt2cAMP‐induced decrease in oxaloacetate and citrate concentration. The fact that lactate enters the pathway between glucose and oxaloacetate indicated that the site of inhibition by Bt, cAMP lies above pyruvate in the glycolytic pathway. Based on these and previous results it appears that, by inhibiting glycolysis, cAMP exerts long‐range effects on two major pathways in the chick hepatocyte, ketogenesis and fatty acid synthesis.
R‐Loop Mapping of Calf‐Thymus RNA Polymerase II. Transcripts in vitro on Restriction Fragments of Adenovirus 2 DNA
Authors: Spindler Ernst , Stein Hans
Published: March 2005

Nascent RNA, synthesized by calf thymus RNA polymerase II on the restriction endonuclease EcoRI fragment A and BamHI fragment B of adenovirus 2 DNA, was rehybridized to the template strand under conditions allowing transcription R‐loop formation. Hybrids, visualized by electron microscopy, were found in looped and in branched configurations, the former being abundant, with an average loop number of roughly four per EcoRI fragment A and three per BamHI fragment B. Frequency distributions of transcription R‐loops, synthesized on identical restriction fragments for different periods of time, showed similar patterns, thus pointing to a nonrandom type of transcription. A comparison of the frequency distribution of R‐loops seen on the two overlapping restriction fragments reveals reasonable coincidence in the loop pattern in one out of the four possible fragment orientations, permitting correlation of the transcription R‐loop pattern with the physical map of the adenovirus 2 DNA. Comparison of start sites tentatively mapped in vitro with the known promoters for RNA polymerase II in vivo suggests that a major transcription site in vitro is located in close proximity to, or identical with, two promoters in vivo at 16.1 and 16.5 map units
The Effect of pH and d‐Glycerate 2,3‐Bisphosphate on the O2 Equilibrium of Norma1 and SH(β93)‐Modified Human Hemoglobin
Authors: ANTONINI Eraldo , CONDÒ Saverio G. , GIARDINA Bruno , IOPPOLO Carmela , BERTOLLINI Albcrto
Published: March 2005

The present paper reports data for the effect of pH and d‐glycerate 2,3‐bisphosphate (d‐glycerate‐2,3‐P2) on the oxygen equilibrium of norma1 and SH(β93)‐modified human hemoglobin. At sufficiently high d‐glycerate‐2,3‐P2 concentrations, both oxy and deoxy forms of HbA are saturated with the organic phosphate at a1l pH va1ues between 6 and 9. Furthermore the difference in the affinity for d‐glycerate‐2,3‐P2 between deoxy and oxy hemoglobin remains constant with pH, implying that the pK va1ues of the Bohr effect groups in deoxy and oxyhemoglobin are not affected by the presence of d‐glycerate‐2,3‐P2 on the hemoglobin molecule.

In the hemoglobins modified in position β93, the difference in affinity between deoxy and oxy hemoglobin for d‐glycerate‐2,3‐P2 decreases with decrease in pH due mainly to a decrease in the affinity of the deoxy form for d‐glycerate‐2,3‐P2. The effect of the chemica1 modification appears to be primarily a change in pK of a Bohr group in deoxy hemoglobin which is especia1ly pronounced in the presence of phosphates.
The Mechanisms of Action of cAMP
Authors: VAN OOL Peter J. J. M. , BUCK Henk M.
Published: March 2005

Quantum chemical calculations were performed on the formation of intermediates with trigonal bipyramidal (TBP) configurations in the hydrolysis of adenosine 3′,5′‐monophosphate (cAMP) with phosphodiesterases and the activation of protein kinases by cAMP. The results show that in the reaction sequence concerning the hydrolysis of cAMP with phosphodiesterase the TBP intermediate must possess an equatorial‐apical cyclic phosphate ring with the 3′‐oxygen atom in the apical position. This could be an additional reason for the sensitivity of the 3′ position in cAMP towards modifications in comparison with the 5′position. According to the calculations, a mechanistic model is presented for the enzymatic hydrolysis of cAMP with the involvement of a covalently bonded enzyme‐nucleotide intermediate. Also a model is offered for the activation of protein kinase by cAMP. The activation of protein kinase is assumed to proceed via diequatorial‐ring‐positioned TBP intermediates resulting in the formation of a covalent bond between cAMP and the protein kinase with retention of the cyclic phosphate ring. It seems likely that the enzyme‐nucleotide intermediate enforces a conformational change in the enzyme, which causes the dissociation of the regulatory and catalytic subunit of the protein kinase, necessary for a physiological response.
Studies on the Secondary Structure of Wheat 5.8 S rRNA
Authors: WILDEMAN Alan G. , NAZAR Ross N.
Published: March 2005

The nucleotide sequence of wheat (Triticum aestivum) 5.8‐S ribosomal RNA has been re‐examined using partial chemical degradation with high‐temperature sequencing gels and oligonucleotide analysis. The results clarify previously ambiguous residues and add two additional nucleotides, G127and G135, to the sequence. Estimates of the secondary structure suggest that 5.8–S rRNAs of higher plants differ from previous examples in having more open G t C–rich and A +U–rich stems. S1 ribonuclease digestion was used to probe this secondary structure;the data generally support the ‘burp gun’ model proposed for all 5.8–S rRNAs but are also consistent with a more open A +U–rich stem. Diethyl pyrocarbonate reactivity was used to probe the topography of this RNA in wheat ribosomes. The results indicate that the G +C–rich and A +U–rich stems are accessible to chemical modification in the wheat ribosome and suggest that the A +U–rich stem undergoes a significant conformational change when the molecule associates into ribosomes.
Isolation and Some Properties of the Restriction Endonuclease BcnI from Bacillus centrosporus
Authors: PETRUŠYTĖ Marytė , JANULAITIS Arvydas
Published: March 2005

A specific type–II restriction endonuclease BcnI from Bacillus cenfrosporus has been purified to electro–phoretic homogeneity in three chromatographic steps. Around 15 μg of such a preparation can be isolated from 1g of the cell paste. The yield of the enzyme is higher than that of any type–II restriction endonuclease so far reported.

The molecular weight of the enzyme determined by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate equals 27 500 and 28000 respectively.

The activity of the restriction endonuclease is maximal at pH 9.2 and 40–45 °C. The optimal magnesium concentration was estimated to be 7.5 mM. The activity of BcnI may also be observed in the presence of Co2+, Mn2+, Ni2+ and Zn2+ but it is markedly less than in the presence of Mg2+.
Prediction of Secondary Structura1 Elements in the Phosphatidylcholine–Transfer Protein from Bovine Liver
Authors: AKEROYD Robert , LENSTRA Johannes A. , WESTERMAN Jan , VRIEND Gerrit , WIRTZ Karel W. A. , VAN DEENEN Laurens L. M.
Published: March 2005

Secondary structural elements of the phosphatidylcholine–transfer protein from bovine liver have been predicted from its primary structure with the aid of two computerized methods. The predicted α–helix and β–strand content have been compared with the values derived from circular dichroism spectra. The hydrophobicity profile (Rose plot) of the protein indicated that the supposed lipid‐binding site occurs in the most hydrophobic region. The predicted secondary structural elements have been folded in a tentative model of the protein molecule according to its hydrophobicity profile.
Extraction of Mitochondrial Protein‐Lipid Complexes into Organic Solvents : An Approach to Study the Interaction between the ATPase and the Mitochondrial ATPase‐Inhibitor Protein
Authors: DARSZON Alberto , GÒMEZ‐PUYOU Armando
Published: March 2005

Protein‐lipid complexes were transferred directly from mitochondria and submitochondrial particles into hexane and ether. The protein‐lipid residue left after solvent removal from these extracts was used to form liposomes which display low‐temperature‐resistant ATPase activity. Centrifugation experiments indicate that the ATPase activity is associated to the vesicles. Most of the F1‐ATPases appear to be accessible to the external water phase of the liposomes. The ATPase activity of these particles was insensitive to dicyclohexylcarbodiimide and oligomycin. Incubation of these vesicles at room temperature activated (4–10‐fold) the ATPase through a process that is partially sensitive to phenylmethylsulfonyl fluoride. The results with purified ATPase‐inhibitor protein and (F1‐ATPase)‐inhibitor complex indicate that the activation process in the liposomes is due to the abolition of the inhibitory action of the inhibitor protein bound to a large fraction of the extracted ATPases. Liposomes prepared from hexane extracts obtained from submitochondrial particles having different levels of ATPase activity displayed an activation ratio which correlated with the number of ATPases that are inhibited by the inhibitor protein in the submitochondrial particles. The extraction of mitochondrial ATPase and its incorporation into liposomes followed by activity measurements may be used to judge the number of ATPases that in a given preparation contain the inhibitor protein in its inhibiting site.
Modification of the Rapidly Reacting Thiols of Atrial and Ventricular Myosins
Authors: SRIVASTAVA Sudhir , WIKMAN‐COFFELT Joan
Published: March 2005

Environmental variances were noted around the rapidly reacting SH‐1 thiol of ventricular versus atrial myosin, based on electron paramagnetic resonance studies. Further studies, in which either SH‐1 or SH‐1 +SH‐2 thiols were modified with N‐ethylmaleimide, indicated the importance of the SH‐2 moiety of both isozymes for gcneration of tension, when analyzed as synthetic actomyosin threads. Comparative EPR studies showed that the spin label was more strongly immobilized when complexed to ventricular SH‐1 thiol as compared to when it was complexed to atrial myosin. Likewise, addition of PP1, ATP or ADP created a greater mobility in the spin label when added to ventricular spin‐labeled myosin as compared to that of atrial myosin. Comparative studies of spin‐labeled actomyosin versus myosin analyzed at different EPR power settings also demonstrated disparaties surrounding the SH‐1 thiol between the two myosin isozymes.
Degradation of Band‐3 Glycoprotein in vitro by a Protease Isolated from Human Erythrocyte Membranes
Authors: GOLOVTCHENKO‐MATSUMOTO Anne Marianne , MATSUMOTO Isamu , OSAWA Toshiaki
Published: March 2005

The use of soybean‐trypsin‐inhibitor‐Sepharose‐4B to purify a protease present in human erythrocyte membranes is described. The fraction bound in the presence of calcium to the affinity adsorbent is active on band‐3 glycoprotein in a non‐ionic detergent solulion at neutral pH. Band‐3 glycoprotein is degraded into components having the mobilities of the proteins of bands 4.5, 7 and of lower molecular weights. When calcium is omittcd from the membrane extract, an inactive form of this enzyme can be purified. By DEAE‐cellulose chromatography this inactive form can be convcrted into the active form, presumably by dissociation of an enzymeinhibitor complex.