bluuebunny/crossref_metadata_2025 huggingface dataset:
Composition of cytochrome P‐450 isozymes from hepatic microsomes of C57BL/6 and DBA/2 mice assessed by warfarin metabolism, immunoinhibition, and immunoelectrophoresis with anti‐(rat cytochrome P‐450)
Authors: KAMINSKY Laurence S. , DANNAN Ghazi A. , GUENGERICH F. Peter
Published: March 2005

The composition of isozymes of hepatic microsomal cytochrome P‐450 (P‐450) from C57BL/6(B6) and DBA/2(D2) inbred strains of mice, uninduced and treated with phenobarbital (PB), pregnenolone‐16α‐carbonitrile (PCN), and β‐naphthoflavone (BNF), were assessed. The assessment was based on comparisons of the regioselectivity and stereoselectivity of warfarin metabolism, inhibition of metabolism by antibodies raised against highly purified rat P‐450 isozymes (the specificity of these antibodies in inhibiting rat microsomal metabolism of warfarin was also determined), and by immunoelectrophoresis using the anti‐(rat P‐450). Isozymes were considered to be the same or very similar if they exhibited the same substrate specificity, antigenicity. and molecular weight. Untreated D2 and B6 mice had similar‐compositions of P‐450 isozymes which were independent of the Ah receptor. Both strains contained an isozyme very similar to rat P‐450UT‐A. PB or PCN induction of the two mouse strains proceeded independently of the Ah receptor and yielded isozymes which are the same as or very similar to the rat P‐450UT‐A, P‐450PB‐B, P‐450PB‐C, and P‐450PB/PCN‐E isozymes. D2 and B6 mice had similar isozyme compositions when treated with PB or PCN, but isozyme compositions varied in differently induced mice. BNF treatment of the D2 mice was without effect, but the B6 mice produced an isozyme(s) which was different from rat P‐450BNF‐B, the major BNF‐induced isozyme in rat liver.
Solvent isotope effects on the glucokinase reaction
Authors: POLLARD‐KNIGHT Denise , CORNISH‐BOWDEN Athel
Published: March 2005

The solvent isotope effects on the reaction catalysed by rat‐liver glucokinase have been studied. At low concentrations of glucose and high concentrations of MgATP2− there is an inverse solvent isotope effect of 3.5. At high glucose concentrations there is a normal solvent isotope effect of 1.3. In 1H2O there is positive co‐operativity with respect to glucose [Storer, A. C. and Cornish‐Bowden, A. (1976) Biochem. J. 159, 7–14], but this is changed to negative co‐operativity in 2H2O. The half‐saturation points for both glucose and MgATP2− are decreased in 2H2O compared with those in 1H2O. Explanations of these effects in terms of the mnemonical model proposed by Storer and Cornish‐Bowden [Biochem. J. 65, 61–69 (1977)] were considered in computer simulation. Two interpretations could account for the results, either a decrease in the rate of interconversion of the two forms of free enzyme postulated in the model, or an increase in the affinity for glucose of the enzyme form with the lower affinity in 1H2O. The results of a proton‐inventory analysis were consistent with either of these interpretations. The solvent isotope effects thus provide additional evidence for the mnemonical model as an explanation of glucokinase co‐operativity.
Surface properties of bacterial sulfhydryl‐activated cytolytic toxins
Authors: ALOUF Joseph E. , GEOFFROY Christiane , PATTUS Franc , VERGER Robert
Published: March 2005

Sulfhydryl‐activated cytolysins are a group of bacterial protein toxins which, in the reduced state, lyse eukaryotic cells by disruption of the cytoplasmic membrane. Cell surface cholesterol is thought to be the target of the toxins. In the present work, the monolayer technique was used to investigate the interaction of four SH‐activated toxins (streptolysin 0, alveolysin, perfringolysin 0, pneumolysin) with various lipid films as a model for studying toxin‐induced membrane disruption. A surface pressure increase up to very high values was elicited by reduced toxins (∼ 10nM) on films of cholesterol, other toxin‐binding 3β‐hydroxy‐sterols, thiocholesterol and cholesterol‐phosphatidylcholine mixtures suggesting deformation or penetration of the films. The surface‐active potency of the toxins was of the same order as that of melittin and snake cardiotoxins at similar concentrations, No pressure increase was observed on films made of pure phosphatidylcholine, lanosterol and other sterols lacking the 3β‐OH group. Optimal efficiency was at cholesterol/phosphatidylcholine molar ratio of 1 to 1. The critical pressures for toxin interaction with phosphatidylcholine and cholesterol monolayers were 25mN · m−1and 45mN · m−1respectively. Toxin interaction with phosphatidylcholine [14C]‐cholesterol films did not modify monolayer radioactivity, indicating no cholesterol desorption. No pressure increase was elicited by toxins inactivated by SH‐group reagents, heating or neutralization with antibody. Toxin effect was dependent temperature and pH. The overall potency of the four toxins tested was streptolysin 0>alveolysin ∼ perfringolysin 0>pneumolysin. The monolayer system mimicked in several respects toxin interaction with eukaryotic cells.
Characterization of a novel killer toxin encoded by a double‐stranded linear DNA plasmid of Kluyveromyces lactis
Authors: SUGISAKI Yuji , GUNGE Norio , SAKAGUCHI Kenji , YAMASAKI Makari , TAMURA Gakuzo
Published: March 2005

A novel killer toxin, encoded by a double‐stranded linear DNA plasmid pGK 1–1 (5.4 MDa) in Kluyveromyces lactis IFO 1267 was purified 320000‐fold from the culture broth of yeast. The toxin was obtained in an electrophoretically homogeneous state with a yield of 24% by hydroxyapatite column chromatography, chromatofocusing and polyacrylamide gel electrophoresis. The purified toxin was dissociated into two subunits with molecular masses of 27 kDa and above 80 kDa, as estimated by Laemmli's sodium dodecylsulfate gel electrophoresis; the exact composition ratio of the two subunits remains unestablished. The isoelectric point was between 4.4 and 4.8. As compared with the reported narrow pH range of action and instability of k1 killer toxin encoded by a double‐stranded RNA plasmid of Saccharomyces cerevisiae, the K. Lactis toxin was effective with sensitive strains of S. cerevisiae in a relatively wider pH range between 4 and 8; it was stable for several months at pH 6.0 when stored below ‐20 °C. In contrast to the simple protein nature of the k1 killer toxin with a molecular mass of 11.47 kDa, the K. lactis toxin maintained a mannoprotein nature, as it was absorbed by a ConA‐Sepharose column and eluted by ethyl α‐d‐mannoside. The growth inhibitory activity of K. lactis toxin was enhanced 2‐35‐fold by the presence of 4–60 % glycerol.
A high‐resolution proton nuclear‐magnetic‐resonance investigation of carp hemoglobin
Authors: DALVIT Claudio , MIURA Shigetoshi , DE YOUNG Alice , NOBLE Robert W. , CERDONIO Massimo , HO Chien
Published: March 2005

The high‐resolution proton nuclear magnetic resonance spectra of carp hemoglobin have been compared to those of human normal adult hemoglobin. Carp deoxy and carbonmonoxy hemoglobins in the deoxy‐type quaternary state exhibit two downfield exchangeable proton resonances as compared to four seen in human normal adult deoxyhemoglobin. This suggests that two of the hydrogen bonds present in human normal adult deoxyhemoglobin are absent or occur in very different environments in carp hemoglobin. One of the exchangeable proton resonances of carp hemoglobin, while present in the deoxy‐type quaternary state of the carbonmonoxy and deoxy derivatives, is absent in the oxy‐type quaternary state of both, in agreement with the assignments of these quaternary structures by other methods. The ring‐current‐shifted proton resonances (sensitive tertiary structural markers) of carp carbonmonoxyhemoglobin are substantially different from those of human normal adult hemoglobin. The aromatic proton resonance region of carp hemoglobin has fewer resonances than that of human normal adult hemoglobin, consistent with its much reduced histidine content. The hyperfine‐shifted proximal histidyl NH‐exchangeable proton resonances of carp hemoglobin suggest that during the transition from the oxy to the deoxy quaternary structure, there is a greater alteration in the heme pocket of one type of subunits (presumably the β chain) than that in the other subunit. The present results suggest that there are differences in both tertiary and quaternary structures between carp and human normal adult hemoglobins which could contribute to the great differences in the functional properties between these two proteins.
Purification and subunit structure of RNA polymerase II from different stages of Artemia development
Authors: CRUCES Jesus , DIAZ Victor , QUINTANILLA Miguel , RENART Jaime , SEBASTIAN Jesús
Published: March 2005

RNA polymerase II has been purified from different stages of Artemia development: cryptobiotic gastrulae, developing embryos and nauplii. RNA polymerase from dormant and developing embryos consists of 12 subunits of 205000, 140000, 32500, 25600, 22000, 19400, 18000, 16800, 147000, 14000, 12700 and 11500 daltons, as determined by dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme from larvae contains the same polypeptides except for the higher‐molecular‐mass component: the larval enzyme has a sununit of 172 kDa instead of the 205‐kDa subunit present in the enzyme purified from embryos. Incubation of embryonic RNA polymerase II with larval extracts results in the conversion of the 205‐kDa subunit into the 172‐kDa one. This conversion is inhibited by soybean trypsin inhibitor, a compound which inhibits three proteases induced during Artemia early larval development. RNA polymerase II purified from a mixture of embryos and larvae contain only the 172‐kDa subunit. Our results indicate that the 172‐kDa subunit present in RNA polymerase II purified from larvae is produced by proteolysis of the 205‐kDa subunit during the extraction and purification of the enzyme and therefore that Artemia has only one class of RNA polymerase II.
Dissociation of ribulose‐1,5‐bisphosphate carboxylase/oxygenase from spinach by urea
Authors: VOORDOUW Gerrit , van der VIES Saskia M. , BOUWMEISTER Peter Pan
Published: March 2005

The dissociation of d‐ribulose‐1,5‐bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques.

Gel chromatography using Sephadex G‐100 indicates the quantitative dissociation of the small subunit S from the complex at 3–4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2‐mercaptoethanol). The dissociated S is monomeric.

Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3–4 M urea sediments with s20,w, = 15.0 S, whereas the intact enzyme (L8S8) sediments with s20,w= 17.7 S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3–4 M urea can thus be represented by L8S8→ L8+ 8 S. At urea concentrations c > 5 M the L8 core dissociates into monomeric, unfolded large subunits.

A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme.

Studies of the circular dichroism of d‐ribulose‐1,5‐bisphosphate carboxylase in the wavelength region 200–260 nm indicate two conformational transitions. The first one ([θ]220 from ‐8000 to ‐3500 deg cm2 dmol−1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([θ]220 from ‐3500 to ‐1200 deg cm2 dmol−1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([θ]220 from ‐8000 to ‐1200 deg cm2 dmol−1) is observed for the activated enzyme.

From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach d‐ribulose‐1,5‐bisphosphate carboxylase: the L‐S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.
Studies in bile salt solutions
Authors: O'CONNOR Charmian J. , WALLACE Robert G.
Published: March 2005

The pseudo‐first‐order rate constants of hydrolysis of p‐nitrophenylacetate, catalyzed by human milk lipase, have been measured in solutions of 0.01 mol dm−3 Bistris(2‐[bis(2‐hydroxyethyl)amino]‐2‐(hydroxymethyl)‐propane‐1,3‐diol) buffer at 310.5 K, containing a range of concentrations of sodium taurocholate and sodium cholate, at pH 8.00 and of sodium cholate at pH 6.5. The effect of pH on the activity of the enzyme has been investigated and the stimulation factors of taurocholate and cholate ions and of cholic acid have been calculated to be equal to 5.3, 3.7 and 10.7, respectively. The essential residues for catalytic activity of the enzyme have ionization constants equal to 6.45–6.46 for pK1 and 8.33–8.40 for pK2. The former value is attributed to the presence of a histidine imidazolium group but the identity of the residue leading to pK2is not proven.
Isolation of active and inactive forms of isocitrate dehydrogenase from Escherichia coli ML308
Authors: BORTHWICK Andrew C. , HOLMS W. Henry , NIMMO Hugh G.
Published: March 2005

In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P. M. and Holms, W. H. (1975) J. Gen. Microbiol. 87, 37–51].

The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion‐red‐Sepharose, from which the enzyme was specifically eluted with NADP+.

Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion‐red‐Sepharose and was essentially inactive; this form could be resolved from the active form by non‐denaturing gel electrophoresis. The other form was specifically eluted from procion‐red‐Sepharose and was partially active; analysis of this form by non‐denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms.

The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of one‐dimensional peptide mapping.

Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non‐denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two‐dimensional peptide mapping showed that both forms were dimers of identical subunits.

The active form of the enzyme contained no detectable alkali‐labile phosphate, the inactive form contained 0.8 molecule/subunit and the partially active form contained an intermediate amount.

The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal).

The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed.
Receptor modulating properties of an antibody directed against the epidermal growth factor receptor
Authors: DAS Manjusri , KNOWLES Barbara , BISWAS Rupamanjari , BISHAYEE Subal
Published: March 2005

A murine antiserum with specificity for the human epidermal growth factor (EGF) receptor was used to investigate EGF receptor function. The IgG fraction of this antiserum displayed no EGF‐like mitogenic activity, even when cross‐linking was ensured by sequential treatment with rabbit anti‐(mouse IgG). The interaction of antibody with solubilized purified EGF receptor was characterized in detail. The binding of 125I‐antibody to the receptor was not blocked by EGF, but the binding of 125I‐EGF to the receptor was blocked by the immune IgG. Scatchard analysis of this reaction revealed a reduction in maximal EGF binding but an enhanced EGF binding affinity. In addition, at low concentrations, the immune IgG was found to enhance receptor kinase activity in the absence of EGF. The enhancement of kinase activity, as measured by receptor phosphorylation, was due to a decreased Km for ATP, and an increased V. These results suggest that the antibody is capable of altering conformations at receptor active sites by binding to non‐active species‐specific epitopes.
Purification and initial characterization of a protein which facilitates assembly of nucleosome‐like structure from mammalian cells
Authors: ISHIMI Yukio , HIROSUMI Jiro , SATO Wakao , SUGASAWA Kaoru , YOKOTA Shoji , HANAOKA Fumio , YAMADA Masa‐atsu
Published: March 2005

A protein, which facilitates assembly of a nucleosome‐like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1983) J. Biochem. (Tokyo) 94, 735–744]. The protein has been purified to approximately 80% from FM3A cells by using histone‐Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53‐kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53‐kDa peptide bound to core histones and formed a 12‐S complex. This complex contained stoichiometrical amounts of the 53‐kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B. H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12‐S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53‐kDa peptide, formation of a 10.5‐S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53‐kDa peptide was detected in the complex.

From above results it is suggested that the 53‐kDa peptide facilitates nucleosome assembly by mediating formation of histone octamer and transferring it to DNA. Rat antibody against the 53‐kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53‐kDa peptide and nucleoplasmin is discussed.
Platelet aggregation: its relation with ADP‐induced fibrinogen binding to platelets and ADP‐related membrane enzyme activities
Authors: LEGRAND Chantal , DUBERNARD Véronique , CAEN Jacques
Published: March 2005

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen‐binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto‐ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP‐induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P < 0.01) was found between the extent of platelet aggregation and the amount of 125I‐fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP‐induced platelet aggregation and fibrinogen binding.

Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP‐induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP‐induced platelet aggregation.
A new method for detection of small modifications in genomic DNA, applied to the human δ‐β globin gene cluster
Authors: CHEBLOUNE Yahia , TRABUCHET Guy , PONCET Didier , COHEN‐SOLAL Michel , FAURE Claudine , VERDIER Géard , NIGON Victor M.
Published: March 2005

Cloned DNA fragments were subcloned in filamentous coliphages fd 103 or M 13; the recombinant single‐stranded DNAs were then used to form hybrids with genomic DNA as well as with complementary recombinant single‐stranded DNA. Hybrids were submitted to S1‐nuclease treatment alone or in combination with restriction enzyme digestions. This method was used to analyze the δ‐β globin gene cluster from the total genomic DNA of a β°‐thalassemic patient. A modification located approximately 530 base pairs upstream from the cap site of the β‐globin gene was detected in only one thalassemic chromosome of this patient. Sequence analysis have shown that the patient was homozygous for a single nucleoside change (dC→dT) which remains undetected by our hybridization method, leading to a codon 39 nonsense mutation; they have demonstrated too that he was heterozygous for the modification mentioned and detected by S1‐nuclease, which corresponds to an additional sequence d(T‐A‐T‐A) in a 52 alternating purine‐pyrimidine run, leading to a complex change from d[(A‐T)7(T)7] to d[(A‐T)11(T)3].
Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat‐brain synaptosomes
Authors: MURAYAMA Satoru , SYUTO Bunei , OGUMA Keiji , IIDA Hiroo , KUBO Shuichiro
Published: March 2005

Botulinum type D neurotoxin was purified 950‐fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 × 107 mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di‐chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits.

Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody.

The binding of type D toxin to rat brain synaptosomes was examined by use of 125I‐labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.
Poly(ADP‐ribose) polymerase is a zinc metalloenzyme
Authors: ZAHRADKA Peter , EBISUZAKI Kaney
Published: March 2005

Purified poly(ADP‐ribose) polymerase was inhibited by 1,10‐phenanthroline at pH <8. This inhibition and the inhibition by other chelating agents suggested that this enzyme was a metalloprotein. Atomic absorption spectroscopy showed the presence of one atom of zinc per protein molecule. Dialysis of the enzyme against buffers containing 1,10‐phenanthroline resulted in the loss of activity and the removal of zinc from the enzyme. Initial rate kinetics snowed that 1,10‐phenanthroline was non‐competitive with NAD+ and competitive with DNA. The binding of DNA to the enzyme was unaffected by the inhibitor. These results suggest that a metal‐containing site is involved as part of the interaction of DNA and poly(ADP‐ribose) polymerase.
Isolation and properties of 5‐aminolevulinate synthase from the yeast Saccharomyces cerevisiae
Authors: VOLLAND Christiane , FELIX Françoise
Published: March 2005

5‐Aminolevulinate synthase from yeast mitochondria has been purified to homogeneity for the first time. By using affinity chromatography on agarose‐hexane‐CoA, gel filtration and DEAE‐Sepharose chromatography, the enzyme was purified about 7000‐fold with an overall yield of 40%. The specific activity of the final preparation was 39000 nmol of 5‐aminolevulinate h−1 mg−1 of protein at 30 C. As judged by gel filtration, polyacrylamide gradient gel and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the enzyme appeared to be composed of two identical subunits of a relative molecular mass of 53000. Electrophoresis of sodium‐dodecyl‐sulfate‐solubilized yeast homogenate followed by immune replica analysis showed that the value of 53000 is the Mr of a non‐degraded form. The purified enzyme had an isoelectric point of 5.3 and a pH optimum of 7.4. Pyridoxal 5′‐phosphate has been shown to be an essential cofactor. The enzyme activity was sensitive to thiol blocking reagents. Hemin, but not heme, inhibited the activity of the purified enzyme.
Pressure dependence of thermolysin catalysis
Authors: FUKUDA Mitsuhiro , KUNUGI Shigeru
Published: March 2005

A comparison of the pressure and temperature dependences of the catalytic reaction of thermolysin, a thermostable neutral protease from Bacillus thermoproteolyticus, with those of a non‐thermostable neutral protease from Bacillus subtilis revealed a distinct difference in Km values of these enzymes for 3‐(2‐furyl)acryloyl‐blocked dipeptide and tripeptide substrates, but not for kcat. Namely, the volume changes for the binding process (ΔV) for these substrates and several competitive inhibitors were –20––30 ml/mol for thermolysin and nearly 0 ml/mol for the non‐thermostable neutral protease. The enthalpy and entropy changes for the binding process were negative for thermolysin, but positive for the latter enzyme. The activation volumes (ΔV≠) for the kcat process were 25–35 ml/mol for both proteases, and activation enthalpy and entropy showed no significant difference between the two enzymes. The characteristic difference in the pressure and the temperature dependences seen for the binding process is discussed in relation to the thermostability of the proteases.
Binding of adriamycin‐Fe3+ complex to membrane phospholipids
Authors: DEMANT Erland J. F.
Published: March 2005

Binding of adriamycin‐Fe3+ complex to phospholipids has been examined by phase partitioning in a hexane‐water system. Formation of a stable ternary adriamycin‐Fe3+‐lipid complex with the negatively charged phospholipids cardiolipin and phosphatidylglycerol (both isolated from Escherichia coli) and synthetic dioleoyl glycerophosphoglycerol is demonstrated.

Binding of adriamycin‐Fe3+ complex to phospholipid bilayers was assessed by incubation of the complex with aqueous phospholipid dispersions prepared from synthetic dimyristoyl glycerophosphocholine, E. coli phosphatidylethanolamine or E. coli cardiolipin and from mixtures of these lipids. Binding of the complex to the phospholipids took place with an affinity for cardiolipin > phosphatidylethanolamine > phosphatidylcholine. The ternary adriamycin‐Fe3+‐ADP complex formed in the presence of 1 mM ADP (ADP in 25‐fold molar excess to adramycin) showed low affinity for binding to phospholipid.
Biological activities of analogues of lipid A based chemically on the revised structural model
Authors: KANEGASAKI Shiro , KOJIMA Yasuhiko , MATSUURA Motohiro , HOMMA J. Yuzuru , YAMAMOTO Akihiro , KUMAZAWA Yoshio , TANAMOTO Ken‐ichi , YASUDA Tatsuji , TSUMITA Toru , IMOTO Masahiro , YOSHIMURA Hiroyuki , YAMAMOTO Michiharu , SHIMAMOTO Tetsuo , KUSUMOTO Shoichi , SHIBA Tesuo
Published: March 2005

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (β‐1,6)‐linked glucosamine disaccharide carrying ester‐bound 3‐hydroxytetradecanoic acids at 3 and 3′ position of reducing and nonreducing glucosamine in addition to amide‐bound 3‐hydroxytetradecanoic acids and glycosidic‐linked and ester‐linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group snowed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.
Antibodies to synthetic peptides as probes of acetylcholine receptor structure
Authors: BARKAS Thomas , JUILLERAT Marcel , KISTLER Joerg , SCHWENDIMANN Bernard , MOODY John
Published: March 2005

Peptides containing the C‐terminus of the alpha‐chain of the nicotinic acetylcholine receptor (nAChR), as deduced from cDNA data, were synthesised and shown to bind to antibodies to denatured nAChR. Conversely, peptide‐specific antibodies bound both to native and to denatured nAChR. Binding was exclusively to the alpha‐chain.

Trypsinization experiments and the use of the unique C‐terminal hexapeptide of the alpha‐chain demonstrated that the proposed C‐terminus does exist on the mature alpha‐chain, and that post‐translational cleavage can be discounted as an explanation of the discrepancy of the molecular masses of the alpha‐chain deduced from SDS gel electrophoresis (40 kDa) and from the DNA sequencing (50 kDa).

Cleavage of the alpha‐chain in the membrane occurs at two closely linked sites, resulting in the formation of a large fragment (approximately 35 kDa) and the remainder of the chain (approximately 9–10 kDa).

No signs of experimental myasthenia gravis were observed in rabbits immunised with C‐terminal peptide coupled to carrier protein.
5‐Enolpyruvylshikimate‐3‐phosphate synthase of Klebsiella pneumoniae
Authors: STEINRÜCKEN Hans Christian , AMRHEIN Nikolaus
Published: March 2005

The shikimate pathway enzyme 5‐enolpyruvylshikimate‐3‐phosphate (EPSP) synthase (3‐phosphoshikimate 1‐carboxyvinyltransferase, EC 2.5.1.19) has been purified to apparent homogeneity from Aerobacter aerogenes, strain 62‐1 (=Klebsiella pneumoniae ATCC 25306). A 3300‐fold purification of the enzyme was achieved by ammonium sulfate fractionation, heat precipitation, chromatography on DEAE‐cellulose, Sephadex G‐75, and cellulose phosphate, and chromatofocusing as the final step. The recovery was 49%.

An apparent relative molecular mass of 32400 was determined by calibrated gel filtration, while a single peptide chain of Mr= 42900 was found by sodium dodecyl sulfate/acrylamide gel electrophoresis. The isoelectric point was determined to be at pH 4.6. Two distinct pH optima (pH 5.4 and 6.8) were observed for the enzyme‐catalyzed formation of EPSP from phosphoenolpyruvate (PEP) and shikimate 3‐phosphate(S3P). For the reverse reaction, the pH optima were 5.6 and 7.6. No evidence for a metal cofactor was found. While the temperature optimum was at 60°C, the activation energies were calculated to be 54.2 kJ/mol for the forward, and 64.1 kJ/mol for the reverse reaction. At low PEP and S3P concentrations, anions acted as activators of EPSP synthase at low concentrations, and as inhibitors at high concentrations.

Non‐linear Lineweaver‐Burk plots were interpreted to result from the activation of EPSP synthase by its anionic substrates. The following dissociation constants were determined for the respective enzyme‐substrate complexes: forward reaction: 43 μM (PEP) and 22 μM (S3P); reverse reaction: 1.3 μM (EPSP) and 2.6 μM (Pi). The kinetic patterns indicate a random sequential mechanism for the forward reaction.
5‐Enolpyruvylshikimate‐3‐phosphate synthase of Klebsiella pneumoniae
Authors: STEINRÜCKEN Hans Christian , AMRHEIN Nikolaus
Published: March 2005

The broad‐spectrum, non‐selective herbicide glyphosate [N‐(phosphonomethyl)glycine] is a potent inhibitor of highly purified 5‐enolpyruvylshikimate‐3‐phosphate (EPSP) synthase (3‐phosphoshikimate 1‐carboxyvinyl‐transferase, EC 2.5.1.19) of Klebsiella pneumoniae. The inhibition is competitive with phosphoenolpyruvate (PEP) with Ki= 1 μM at pH 6.8 and non‐competetive with shikimate 3‐phosphate, EPSP, and inorganic phosphate. Non‐herbicidal analogues of glyphosate, such as aminomethylphosphonic acid, bis‐N‐(phosphonomethyl)glycine and iminodiacetic acid, do not inhibit the enzyme. Inhibition of EPSP synthase by glyphosate strongly increases with increasing pH. Glyphosate protects the enzyme against inactivation by phenylglyoxal, 3‐bromopyruvate, and N‐ethylmaleimide. It is proposed that glyphosate binds to the PEP‐binding site of EPSP synthase as a transition‐state analogue of PEP. Other PEP‐utilizing enzymes were not found to be subject to inhibition by glyphosate.
Enzymatic preparation of an immunostimulant, the disaccharide‐dipeptide, N‐acetyl‐β‐D‐glucosaminyl‐(1→4)‐N‐acetylmuramyl‐L‐alanyl‐D‐isoglutamine, from a bacterial peptidoglycan
Authors: GUINAND Micheline , FRANÇON Alain , VACHERON Marie‐Jeanne , MICHEL Georges
Published: March 2005

The disaccharide‐dipeptide N‐acetyl‐β‐D‐glucosaminyl‐(1→4)‐N‐acetylmuramyl‐L‐alanyl‐D‐isoglutamine has been obtained by an enzymatic degradation of the peptidoglycan of Actinomadura R39. The peptidoglycan was hydrolyzed successively by the three following enzymes: lysozyme, DD‐carboxypeptidase from Streptomyces albus G and γ‐D‐glutamyl‐meso‐diaminopimelate endopeptidase I from Bacillus sphaericus 9602. The by‐products of the last reaction were eliminated by successive ion‐exchange and gel‐permeation chromatographies. Both chemical analysis and mass spectrometry show that the resulting disaccharide‐dipeptide is a pure compound.
Photo‐induced dimerization of anthracene phospholipids for the study of the lateral distribution of lipids in membranes
Authors: de BONY Jacqueline , TOCANNE Jean‐François
Published: March 2005

It is shown that photo‐induced cross‐linking reaction between anthracene‐labelled phospholipids can be used for studying, at a molecular level, their lateral distribution in bilayer structures. A simple and versatile method is proposed. It is based on the property of anthracene to form covalently bound dimers upon irradiation in the near ultraviolet (360 nm) and on the possibility of separating the lipid photo‐dimers from the lipid monomers by thin‐layer chromatography. Identification of the photo‐dimers is easily achieved since, upon illumination at a shorter wavelength (250–280 nm), they partially dissociate to the native monomer molecules.

The feasibility of the method was tested by checking the effects of cations (sodium, calcium) on the homogeneity of 1/1 mixtures of anthracene‐phosphatidylcholine, i.e. 1‐acyl‐2‐[9‐(2‐anthryl)‐nonanoyl]‐sn‐glycero‐3‐phosphocholine (Anthr‐PC) with anthracene‐phosphatidic acid (Anthr‐PA) and with anthracene‐phosphatidylglycerol (Anthr‐PG) in the form of liposomes. These lipids were anthracene‐labelled by acylation of their glycerol backbone at the sn‐2 position with the synthetic 9‐(2‐anthryl)‐nonanoic acid. Data presented indicate a good miscibility of these lipids in the presence of sodium. For each lipid mixture, the lipid heterodimers were clearly identified and, quantitatively, they dominated the lipid homodimers, as expected for a regular distribution of the lipids in the 1/1 mixture. Addition of calcium ions to the lipid suspensions did not alter the miscibility properties of Anthr‐PC and Anthr‐PG. In contrast, calcium triggered a clear‐cut phase separation in the Anthr‐PC/Anthr‐PA mixture as, in this case, only traces of the heterodimer form of the lipids remained observable on the chromatogram. The three anthracene‐phospholipids, pure or mixed together, exhibit a clear‐cut gel‐to‐liquid phase transition which was detectable by fluorescence intensity measurements. The analysis of, the corresponding phase‐transition temperatures confirms, at a ‘macroscopic’ level, the effects of sodium and calcium on the mixing properties of the anthracene phospholipids which were revealed at a ‘microscopic’ level by the dimerization procedure.
The fimA gene encoding the type‐1 fimbrial subunit of Escherichia coli
Authors: KLEMM Per
Published: March 2005

The nucleotide sequence was determined of a region of 1450 base pairs encompassing the fimA gene for the subunit of type 1 fimbriae of Escherichia coli as well as flanking regions containing potential regulator sequences. The ‘translated’ protein contains a 23‐residue signal peptide; the processed fimbrial subunit consists of 158 amino acid residues yielding a relative molecular mass of 15706. The elucidated sequence shows significant homology with those of other E. coli fimbrial proteins.
A study of the interactions between residues in the C‐terminal half of calmodulin by one and two‐dimensional NMR methods and computer modelling
Authors: AULABAUGH Ann , NIEMCZURA Walter P. , BLUNDELL Thomas L. , GIBBONS William A.
Published: March 2005

Assignments of the six sets of aromatic ring protons and four high‐field‐shifted methyl group protons of the C‐terminal fragment of calmodulin, residues 78–148, was achieved by a combination of one and two‐dimensional NMR spectroscopic methods. A full spectral analysis of the aromatic region in terms of chemical shifts and scalar coupling constants was achieved and confirmed by spectral simulation.

A three‐dimensional structural model of the C‐terminal fragment was constructed by interactive computer graphics techniques and combined with nuclear Overhauser enhancements to propose sequence assignments for all aromatic and high‐field‐shifted methyl groups. This computer‐generated three‐dimensional model was generally supported by the fact that it qualitatively accounted for many of the ring‐current‐shifted proton resonances and the intraresidue and interresidue nuclear Overhauser enhancements.
Apolipoproteins as the basis for heterogeneity in high‐density lipoprotein2 and high‐density lipoprotein3
Authors: MARCEL Yves L. , WEECH Philip K. , NGUYEN Thanh‐Dung , MILNE Ross W. , McCONATHY Waller J.
Published: March 2005

A method is described for the isoelectric focusing (IEF) of lipoproteins on thin films of agarose. Within a pH gradient of 4.60–5.30 both high‐density lipoproteins 2 and 3 (HDL2 and HDL3) are resolved into more than 10 fractions which could be stained either for protein or for lipids. The isoelectric focusing patterns for HDL3 and HDL3 are similar although HDL2 appears richer in the more alkaline bands.

Narrow film strips from the IEF separation of HDL2 and HDL3 were interfaced with various agarose plates containing antisera against apolipoproteins apoAI, apoAII and apoCIII either alone or in combination, to provide two‐dimensional IEF immunoelectrophoresis patterns. This technique demonstrated that apoAI and apoAII were present throughout the IEF gel for both subclasses of HDL. It also provided evidence for the existence of lipoproteins containing both apoAI and apoAII and other lipoproteins present in the alkaline region of the gel which contained apoAI but no apoAII. ApoCIII was found mostly in acidic lipoproteins and was not distributed identically in HDL2 and HDL3. The lipoproteins separated by IEF on agarose were also analysed by two‐dimensional IEF‐SDS electrophoresis and the individual apo lipoproteins were identified by reaction with antibodies to apolipoproteins AI, AII, CI, CII, CIII, D, and E. This technique confirmed that in IEF of HDL, apoAI extended throughout the spectrum of lipoproteins whereas apoE was only present in alkaline lipoproteins and apoD was only present in acidic lipoproteins.

IEF on agarose of either HDL2 or HDL3 allowed us to collect eight different fractions, which have the same pi in either lipoprotein class. The apolipoprotein composition of each isolated band was analysed by electroimmunoassays for apolipoproteins AI, AII, CI, CII, CIII, D, and E and the results expressed as the ratio of the measured apolipoprotein to measured apoAI. In both HDL2 and HDL3, acidic lipoprotein fractions were enriched in apoAII, apoCIII and apoCI. ApoCII and apoCIII were not similarly distributed in HDL2 and HDL3 subfractions whereas the apoCI distribution was similar in both classes. Noteworthy in all experiments was the difference in the distributions of apoCI, apoCII, and apoCIII in HDL2 and HDL3, which indicated that the existence of a lipoprotein containing simultaneously CI, CII and CIII can only account for a small fraction of these apolipoproteins.

Therefore these experiments substantiate the theory of the protein basis of HDL heterogeneity and suggest that the majority of apolipoproteins are present in complexes which upon IEF result in lipoprotein fractions of identical pi for both HDL2 or HDL3. In addition, subtle differences in the distribution of apolipoproteins in HDL2 and HDL3 indicate the occurrence of some different lipoprotein species in these two subclasses of HDL.
Regulation of hepatic glutamate metabolism
Authors: HÄUSSINGER Dieter , GEROK Wolfgang
Published: March 2005

1. In isolated perfused rat liver, addition of the oxoanalogues of leucine, isoleucine, methionine and phenylalanine is followed by a rapid and reversible stimulation of glutamate release. This is not observed with the corresponding amino acids or 2‐oxoisovaIerate, 2‐oxoglutarate or oxaloacetate.

2. The increased glutamate release by the liver is accompanied by a decrease in the tissue contents of 2‐oxoglularate and glutamate by about 25% and 50%, respectively.

3. During the metabolism of glutamine, i.e. conditions with elevated tissue glutamate concentrations, 2‐oxoacid‐induced glutamate release is stimulated. In the presence of glutamine (5 mM), 2‐oxoisocaproate, 2‐oxo‐4‐melhylvalerate and 2‐oxo‐4‐methylthiobulyrate were found to be most effective and glutamate release by the liver increased linearly from about 80 nmol g−1 min−1 to 600 nmol g−1 min−1 at increasing 2‐oxoacid concentrations up to 1mM, When glutamate [issue levels were decreased by phenylephrine, stimulation of glutamate release by 2‐oxoisocaproate was markedly diminished.

4. 2‐Oxoacid‐stimulated glutamate release is independent of oxoacid metabolism, indicating that the effect is probably not explained by a 2‐oxoacid/glutamate exchange across glutamate export predominantly occurs in a sodium‐independent way.

5. At low concentrations of 2‐oxoisocaproate (below 0.2 mM), the increased glutamate release was accompanied by a slight inhibition of 14CO2 production from added [14C] glutamate, indicating a simultaneous glutamate uptake and release also under these conditions.

6. Stimulation of glutamate release by 2‐oxoisocaproate is followed by a decreased rate of urea and glutamine synthesis from portal ammonia, as a consequence of, an increased glutamate release.
Cloning and sequencing of a sheep metallothionein cDNA
Authors: PETERSON M. Gregory , LAZDINS Ieva , DANKS David M. , MERCER Julian F. B.
Published: March 2005

A partially purified metallothionein mRNA fraction from copper‐injected sheep liver was used to synthesize double‐stranded cDNA, which was dC‐tailed, annealed to dG‐tailed pBR322 and used to transform Escherichia coli MC1061. Of the 1500 recombinant clones only one gave a positive signal when screened with a mouse metallothionein 1 probe. This clone (pSMT‐1) contained an insert which included the entire coding region of a sheep metallothionein, the whole 3′‐untranslated region, part of the poly(A)‐tail and 25 bases of the 5′‐untranslated region. DNA sequence analysis showed that this sheep metallothionein was very similar to other mammalian metallothioneins except for a threonine to proline change at amino acid 27. The clone also contained a different polyadenylation signal d(A‐G‐T‐A‐A‐A) from that usually found; d(A‐A‐T‐A‐A‐A). Comparison of the DNA sequence of the sheep metallothionein with those of other species revealed an interesting region of homology close to the poly(A) addition signal in the 3′‐untranslated region of the mRNA.
Interaction of calmodulin with muscle phosphofructokinase
Authors: MAYR Georg W.
Published: March 2005

Phosphofructokinase from muscle has been shown to be a calmodulin‐binding protein [Mayr, G. W., and Heilmeyer, L. M. G., Jr (1983) FEBS Lett. 159, 51–57]. Details of the influence of calmodulin on the aggregation state, the conformation and the catalytic properties of phosphofructokinase have been studied by enzymatic and light‐scattering analyses. Calmodulin acts as a Ca2+‐dependent hysteretic inhibitor of the highly active enzyme. At least one mole of calmodulin binds to each protomer of the enzyme, induces a shift from the highly active tetrameric towards an inactive dimeric state and slowly changes the conformation of the dimers. Dissociation of calmodulin from conformationally changed dimers by removal of Ca2+ stops the inactivation. Without a significant regain of catalytic activity large polymers are rapidly formed. For a reactivation, of the inactivated enzyme, calmodulin has to remain associated and the incubation conditions must be changed in a way to allow for a back isomerization and reassociation of dimers. The isomerization reaction is promoted by Mg · ATP, the reassociation reaction most effectively by fructose bisphosphate. A model for the calmodulin‐phosphofructokinase interaction is proposed.
Structure of human‐basement‐membrane (type IV) collagen
Authors: BABEL Wilfred , GLANVILLE Robert W.
Published: March 2005

The complete amino acid sequence of the 914‐residue‐long pepsin fragment α1(IV)95 from the α1 chain of human placental basement membrane (type IV) collagen is presented. This sequence contains 12 interruptions of the collagenous triplet sequence Gly‐Xaa‐Yaa which varied in length from 1 to 11 residues. The distribution of amino acids between the Xaa and Yaa position was similar to that found in interstitial collagens but the extent of proline and lysine hydroxylation differed. Computer comparisons of the α1(IV)95 sequence with those of the interstitial collagen chains did not reveal any homology, whereas a comparison with the partial sequences of mouse tumor and bovine lens capsule α1(IV) showed an approximately 85% identity. The unique sequence characteristics of type IV collagen are discussed in relation to its macromolecular structure and to the interstitial collagens.
Characterization and function of pig intestinal sucrase‐isomaltase and its separate subunits
Authors: RODRIGUEZ Ignacio R. , TARAVEL Francois R. , WHELAN William J.
Published: March 2005

1. Papain‐solubilized pig intestinal sucrase‐isomaltase was purified to homogeneity in a four‐step process with a yield of 50%.

2. The substrate specificities of the two enzyme activities were studied together and separately after inactivation or inhibition of one of the activities.

3. Michaelis constants, maximum velocities and time courses of hydrolysis of several substrates, in particular α‐limit dextrins, were used to characterize this complex of α‐glucosidases.

4. The participation of the enzyme complex in the hydrolysis of α‐limit dextrins and more generally in pathways for starch breakdown in the pig digestive tract is examined and discussed.