bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential distribution of group‐II‐like phospholipase A2 in mononuclear phagocytic cells in rat spleen and liver
Authors: INADA Masami , TOJO Hiromasa , KAWATA Sumio , TARUI Seiichiro , OKAMOTO Mitsuhiro
Published: March 2005

The localization of calcium‐dependent phospholipase A2, (PLA2) immunochemically closely related to the enzyme of the viperid and crotalid type (group II), in cells isolated from rat spleen and liver was examined using a polyclonal antibody directed against rat spleen group II, PLA2 (PLA2M). In isolated spleen cells, the monocyte/macrophage fraction had the highest PLA2 activity (1.28 ± 0.35 · min−1· 106 cells−1) which was almost completely inhibited by the anti‐PLA2M antibody. An immunoblot analysis confirmed the presence of the enzyme in this fraction. An immunocytochemical study revealed that the PLA2 was present in spleen macrophages. In the isolated liver cells, Kupffer cells (0.92 ± 0.22 nmol · min−1· 106 cells−1) contained higher anti‐PLA2M‐antibody‐inhibitable PLA2 activity than parenchymal cells (0.26 ± 0.06 · min−1· 106 cells−1). The immunocytochemical study showed that cells immunopositive with anti PLA2M antibody were Kupffer cells. These results suggest that the mononuclear phagocytic cells in rat spleen and liver have relatively high activity of group‐II‐like PLA2. Subcellular distribution patterns of the anti‐PLA2M‐antibody‐inhibitable phospholipase A2 activity in different cell populations from spleen and liver were compared. A mode of the distribution of the enzyme in the spleen macrophages was essentially similar to that in the spleen lymphocytes. The distribution in Kupffer cells was similar to that in parenchymal cells.
Pumpkin malate synthase
Authors: MORI Hitoshi , TAKEDA‐YOSHIKAWA Yoko , HARA‐NISHIMURA Ikuko , NISHIMURA Mikio
Published: March 2005

A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)‐rich RNA of etiolated pumpkin cotyledons. Determination of the DNA sequence of the 1979‐nucleotide cDNA revealed a 1698‐nucleotide open reading frame that encodes a polypeptide of 64632 Da. The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide‐sequence analyses of fragments generated by acid treatment of the purified enzyme. Northern blot analysis revealed that the probe hybridized to a single 2.3‐kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons. In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively. Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed.
Molecular cloning, expression and nucleotide sequence of the endo‐β‐1,3‐1,4‐d‐glucanase gene from Bacillus licheniformis Predictive structural analyses of the encoded polypeptide
Authors: LLOBERAS Jorge , PEREZ‐PONS Josep A. , QUEROL Enrique
Published: March 2005

A Bacillus licheniformis gene coding for an endo‐β‐1,3‐1,4‐d‐glucanase have been cloned in Escherichia coli and sequenced. The open reading frame contains a sequence of 731 bp, encoding a polypeptide of 243 amino acid residues, with a molecular mass of 27404 Da (24418 Da without the putative signal peptide), which corresponds to the enzyme we had previously isolated and characterized. The signal peptide is functional in E. coli. More than 60% of the endo‐β‐1,3‐1,4‐d‐glucanase activity is extracellular or periplasmic. The polypeptide is highly similar to other reported Bacillus β‐glucanases. Several structural predictive analyses (secondary structure, hydropathic plots, similarity with other related enzymes, etc.) have been performed. From these analyses we assign a tentative three‐functional‐domain structure for the enzyme (signal peptide, substrate binding and catalytic domains) and a putative lysozyme‐like active site.
Thermodynamics and modulation of progesterone microcompartmentation and hydrophobic interaction with cytochrome P450XVII based on quantification of local ligand concentrations in a complex multi‐component system
Authors: KÜHN‐VELTEN W. Nikolaus
Published: March 2005

An approach towards the determination of hydrophobic ligand distribution in endoplasmic reticulum membrane suspensions, and of hydrophobic ligand interaction with membrane‐anchored proteins, based on calculations of local ligand pools, is presented. Rat testicular microsomes containing cytochrome P450XVII (P450XVII) were used as the model system and considered as consisting of three compartments, i.e. membrane lipid phase, aqueous phase and the ligand‐binding protein, P450XVII. Combinations of spectrophotometry, ultracentrifugation and equilibrium dialysis were used to quantify progesterone concentrations in each of the three compartments, as well as partition coefficients, Kp. Since the substrate‐access channel of P450XVII is likely to face the membrane‐lipid phase, corrected spectral dissociation constants, KcorrS, were calculated on the basis of free, i.e. not enzyme‐bound, progesterone concentrations in the membrane compartment. Modulation of individual components and construction of more complex systems demonstrated the validity of this concept for analysis of multicompartment systems. Although ligand distribution was considerably affected by both ligand and membrane concentrations, Kp and KcorrS values were found to be independent of both parameters; Kp values amounted to 1920 and 3120, and KcorrS values amounted to 260 μM and 96 μM at 4°C and 25°C, respectively. Thermodynamic parameters ΔH, ΔS and ΔG were calculated from Van't Hoff plots for progesterone partition into the membrane compartment, and for progesterone binding to P450XVII. Both of these processes were entropy dominated, and free energy changes amounted to about–18 kJ/mol for Kp and–20 kJ/mol for KcorrS. Modification of P450XVII by gonadotropin‐induced down‐regulation, and by addition of a competitive inhibitor (estradiol) had no effect on progesterone partition. Consideration of Kp= 310 for estradiol allowed the determination of a corrected KI= 3.09 mM. Modification of the membrane‐lipid phase by detergents affected progesterone‐P450XVII interaction solely by modulation of Kp; modification of the aqueous phase by addition of bovine serum albumin as a fourth compartment acted solely via additional steroid attraction. This model system therefore stresses the relevance of the local environment of membrane‐bound enzymes or receptors for quantification of their interaction with substrates or ligands.
pH‐dependent mechanism in the catalysis of prolyl endopeptidase from pig muscle
Authors: POLGAR László
Published: March 2005

Prolyl endopeptidase, an enzyme exhibiting high specificity towards the Pro‐Xaa bond, is thought to play an important role in the metabolism of biologically active peptides. (a) It has been found that pig muscle is an appropriate source for the preparation of a reasonable quantity of enzyme. Thus, 1 kg muscle yields 1–1.5 mg enzyme, homogeneous by fast protein liquid chromatography. (b) Bulky reagents reacting with cysteine residues inactivate the enzyme almost completely, whereas the small molecule iodoacetamide only partially inhibits the catalytic activity. This indicates that the thiol group is not essential for catalysis, but it is located at or near the active site. (c) Kinetic analysis has indicated that prolyl endopeptidase is remarkably sensitive to ionic strength. Addition of salts, e.g. sodium chloride, to the reaction mixture, up to 0.5 M, considerably enhances the rate of acylation, suggesting that charged groups of the protein exert significant effects on the catalytic site. (d) This is supported by the doubly sigmoidal character of the pH‐rate profile. The existence of the doubly sigmoidal curve also indicates that the enzyme has two forms, which exhibit different activities and interconvert with changing pH. (e) The low‐pH form displays a significant kinetic deuterium isotopic effect (1.70 and 1.89 in the absence and in the presence of 0.5 M NaCl, respectively), as usually observed in the serine protease catalysis. In contrast, the high‐pH form, which is physiologically operative, however, has practically no kinetic deuterium effect (1.09 and 1.15 in the absence and in the presence of 0.5 M NaCl, respectively). It is concluded that a general base/acid‐catalyzed acylation step is rate‐limiting in the lower pH range, and an isotopically silent step, probably a conformational change preceding acylation, dominates the reaction in the physiological pH range.
Heparinase II from Flavobacterium heparinum
Authors: MOFFAT Colin F. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B.
Published: March 2005

Five chemically modified heparins were derived from native pig mucosal heparin (pig heparin Is). These were de‐N‐sulphated heparin (heparin Ih), N‐acetylheparin (heparin Ia), de‐N/O‐sulphated heparin (heparin IVh), de‐O‐sulphated heparin (heparin IVs) and de‐O‐sulphated N‐acetyl‐heparin (heparin IVa). Their structures were studied by 13C‐NMR spectroscopy at 90.56 MHz. Native heparin and the derivatives were incubated with Flavobacterium heparinase II at 25°C. The progress of degradation was followed by the δA235 and the final composition examined by gel filtration with Bio‐Gel P‐4.

Native heparin (Is) was readily degraded by heparinase II and, with the exception of heparin IVh for which degradation was negligible, the chemically modified derivatives were also degraded. Approximately 90% of the saccharides from heparins Is, Ia, IVs and IVa were disaccharides and tetrasaccharides. For heparin Ih, which was degraded more slowly, the proportion was 65%. Heparins Is, IVs and IVa underwent initial rapid degradation. The digestion of heparin Ia proceeded rapidly after an initial lag phase.

The undegraded polymers produced similar elution profiles from Bio‐Gel P‐4. Following the action of heparinase II on heparins Is, Ia, IVs and IVa, the elution profiles revealed a major peak of disaccharides and minor peaks of higher oligomers. The profile of heparin Ih revealed a greater proportion of intermediate‐molecular‐mass saccharides.

Our results demonstrate a broad specificity for heparinase II. It is capable of lysing both N‐acetylated and N‐sulphated heparins independent of O‐sulphation. Heparinase II will also degrade heparin derivatives that are non‐N‐substituted provided that they are O‐sulphated.
Catalytic properties of phenol carboxylase In vitro study of CO2: 4‐hydroxybenzoate isotope exchange reaction
Authors: LACK Achim , TOMMASI Immacolata , ARESTA Michele , FUCHS Georg
Published: March 2005

Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para‐carboxylation to 4‐hydroxybenzoate (biological Kolbe‐Schmitt synthesis). The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named ‘phenol carboxylase’, catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4‐hydroxybenzoate (specific activity 0.1 μmol 14CO2 incorporated into 4‐hydroxybenzoate × min−1× mg−1 cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from [14C]phenol was not exchanged into 4‐hydroxybenzoate ring positions to a significant extent. The 14CO2 isotope exchange reaction was studied in vitro. The reaction was dependent on the substrates CO2 and 4‐hydroxybenzoate and required K+ and Mn2+. The actual substrate was CO2 rather than HCO3−. The apparent Km values were 1mM dissolved CO2, 0.2 mM 4‐hydroxybenzote, 2 mM K+, and 0.1 mM Mn2+. The cationic cocatalysts could be substituted by ions of similar ionic radius: K+ could be replaced to some extent by Rb+, but not by Li+, Na +, Cs+, or NH4+; Mn2+ could be replaced to some extent by Fe2+ >Mg2+, Co2+, but not by Ni2+, Zn2+, Ca2+, or Cu2+. The exchange reaction was not strictly specific for 4‐hydroxybenzoate, however it required a p‐hydroxyl group: derivatives of 4‐hydroxybenzoate with OH, CH3 or Cl substituents in mposition did react, whereas those with substitutions in the o‐position were inactive or were inhibitory. The enzyme was induced when cells were grown on phenol, but not on 4‐hydroxybenzoate. Comparison of SDS/PAGE protein patterns of cells grown on phenol or 4‐hydroxybenzoate revealed several additional protein bands in phenol‐grown cells. The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed.
The involvement of extracellular calcium in the formation of 5‐lipoxygenase metabolites by human polymorphonuclear leukocytes
Authors: SCHATZ‐MUNDING Monika , HATZELMANN Armin , ULLRICH Volker
Published: March 2005

We have addressed the question why in the presence of a Ca2+ ionophore human polymorphonuclear leukocytes generate leukotrienes in high yields, but in only low amounts after stimulation by receptor agonists like fMLF (fM, formylmethionine), leukotriene B4 or platelet‐activating factor (PAF), although a significant release of intracellular calcium can be measured. Using ionomycin we can show that from the two enzymes involved, phospholipase A2 and 5‐lipoxygenase, the first requires a threshold level of about 350–400 nM calcium whereas 5‐lipoxygenase shows a linear dependence on calcium and saturates at this concentration. Our data indicate that the Ca2+ requirement of phospholipase A2 can only be met by an additional influx of extracellular calcium, whereas 5‐lipoxygenase will operate already at levels provided by intracellular stores. Consequently, the complexing of extracellular calcium by EGTA stops phospholipase A2 activity immediately, whereas added arachidonate can be still adequately metabolized by intracellular Ca2+ release triggered by fMLF or PAF. Interestingly, PAF shows a stronger extracellular component in its Ca2+ transient than fMLF, and also generates more 5‐lipoxygenase metabolites. However, a clear correlation between the amount of 5‐lipoxygenase metabolites and the extracellular Ca2+ signal was lacking, since maximal activity was achieved before the bulk of the extracellular calcium was monitored. Ca2+ influx after PAF stimulation could be blocked after 2 min by EGTA, but a further increase in the formation of 5‐lipoxygenase metabolites was observed. In contrast ionomycin‐elicited 5‐lipoxygenase activity could be stopped at any time shortly after EGTA addition.
Mapping the functional domains of the eukaryotic elongation factor 1βγ
Authors: DAMME Henk van , AMONS Reinout , JANSSEN George , MÖLLER Wim
Published: March 2005

The functional domains of the eukaryotic elongation factor (EF) 1βγ have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non‐denaturing conditions and antibodies against EF‐1β and EF‐1γ. By means of limited proteolysis, it was possible to obtain large fragments of EF‐1β. In contrast to amino‐terminal fragments, those derived from the carboxy‐terminal part of EF‐1β were still active in enhancing the guanine nucleotide exchange of GDP bound to EF‐1α. With the same technique of limited proteolysis, it was possible to isolate a trypsin‐resistant core from EF‐1βγ containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF‐1β and two monoclonal antibodies against EF‐1γ were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino‐terminal and another on the carboxy‐terminal half of EF‐1γ. The antiserum against EF‐1β and one of the monoclonal antibodies (mAb 36E5), which recognized the amino‐terminal half of EF‐1γ, reacted with this trypsin‐resistant core. We conclude that the amino‐terminal halves of both EF‐1β and EF‐1γ are firmly attached to each other, and that the carboxy‐terminal part of EF‐1β interacts with EF‐1α.
A study on apoenzyme from Rhodotorula gracilis D‐amino acid oxidase
Authors: CASALIN Paola , POLLEGIONI Loredano , CURTI Bruno , SIMONETTA Mirella PILONE
Published: March 2005

The apoenzyme of d‐amino acid oxidase from Rhodotorula gracilis was obtained at pH 7.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M potassium phosphate, 0.3 mM EDTA, 5 mM 2‐mercaptoethanol and 20% glycerol. To recover a reconstitutable and highly stable apoprotein, it is essential that phosphate ions and glycerol be present at high concentrations. Apo‐d‐amino acid oxidase is entirely present as a monomeric protein, while the reconstituted holoenzyme is a dimer of 79 kDa. The equilibrium binding of FAD to apoprotein was measured from the quenching of flavin fluorescence and by differential spectroscopy: a Kd of 2.0 × 10−8M was calculated. The kinetics of formation of the apoprotein‐FAD complex were studied by the quenching of protein and flavin fluorescence, by differential spectroscopy and by activity measurements. In all cases a two‐stage process was shown to be present with a fairly rapid first phase, followed by a slow secondary change which represents only 4–6% of the total recombination process. In no conditions was a lag in the recovery of maximum catalytic activity observed. The process of FAD binding to yeast d‐amino acid oxidase appears to be of the type Apo + FAD ⇋ 2 holoenzyme, even though the existence of a transient intermediate not detectable under our conditions cannot be ruled out.
Isolation and characterization of a membrane glycoprotein from human brain with sequence similarities to cell adhesion proteins from chicken and mouse
Authors: BERGLUND Erik , STIGBRAND Torgny , CARLSSON Sven R.
Published: March 2005

We previously described the production of monoclonal antibodies against a preparation of membrane glyco‐proteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809–815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135).

Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N‐glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments.

Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins.
Interaction of Zn2+ with the bovine‐heart mitochondrial bc1 complex
Authors: LORUSSO Michele , COCCO Tiziana , SARDANELLI Anna Maria , MINUTO Michele , BONOMI Francesco , PAPA Sergio
Published: March 2005

A study is presented of the effect of Zn2+ on the enzymatic properties of the bovine‐heart cytochrome‐bc1 complex. Micromolar concentrations of Zn2+ reversibly inhibit the cytochrome‐c reductase activity of either the cholate‐solubilized or liposome‐reconstituted complex. Kinetic analysis of the redox reactions of the cytochromes indicate that Zn2+ affects the activity of the complex at the quinol oxidation site. The following have been determined: (a) Zn2+ inhibits the pre‐steady‐state reduction of cytochrome c1 by duroquinol either in the absence or in the presence of antimycin, (b) it does not inhibit the reduction of b cytochromes in the absence of antimycin or in the presence of myxothiazol, (c) it inhibits cytochrome‐b reduction in the presence of antimycin. Furthermore Zn2+ inhibits the antimycin‐promoted oxidant‐induced extrareduction of b cytochromes.

Addition of Zn2+ to reduced bc1 complex causes a red shift in the absorption spectrum of cytochrome b566 and a substantial decrease in the signal intensity of the EPR spectrum of the Fe‐S protein. This is interpreted as an interaction of Zn2+ with the 2Fe–2S‐cluster region of the Fe‐S protein, thus giving rise to inhibition of the reductase activity and of the antimycin‐insensitive reduction route of b cytochromes.

A Scatchard‐plot of 65Zn2+ binding to the native isolated complex gave a straight line from which a value of three binding sites and a single dissociation constant of 3 × 10−6 M can be calculated, which is practically equal to the concentration causing 50% inhibition of electron flow.
Metal selectivity of clusters in rabbit liver metallothionein
Authors: GOOD Meinrad , HOLLENSTEIN Roger , VAŠÁK Milan
Published: March 2005

In mammalian metallothioneins the metals are organized in two adamantane‐type clusters with three and four metal ions which are tetrahedrally coordinated by thiolate ligands. The metal selectivity of the metal‐thiolate clusters in rabbit liver metallothionein has been studied by offering two ions, i.e. Co(II)/Cd(II), Zn(II)/Cd(II) or Co(II)/Zn(II), to the metal‐free protein. The heterogeneous metal complexes thus formed were characterized by electronic absorption, magnetic circular dichroism, 113Cd‐NMR and EPR spectroscopy. In the case of Co/Cd‐metallothionein, homometallic cluster occupation occurs, with the Cd(II) ions bound exclusively to the four‐metal cluster. In contrast, heterometallic clusters were formed for both Zn/Cd‐ and Co/Zn‐metallothionein. Based on evidence from corresponding inorganic structures of adamantane metal‐thiolate cages, it is suggested that the major factor governing the cluster type is the protein structure perturbation due to the cluster volume variations. Thus, while metal thiolate affinities are important in the folding process, size‐match selectivity is the dominant factor in the metal‐loaded protein.
Crystalline green 5‐hydroxyvaleryl‐CoA dehydratase from Clostridium aminovalericum
Authors: EIKMANNS Ulrich , BUCKEL Wolfgang
Published: March 2005

A green enzyme from Clostridium aminovalericum with valeryl‐CoA dehydrogenase activity was purified to homogeneity (169 ± 3 kDa) and crystallized. By SDS/PAGE, one type of subunit (42 kDa) was detected indicating a homotetrameric structure. The unusual ultraviolet/visible spectrum of the green enzyme (maxima at 394 nm, 438 nm and 715 nm) was converted to a normal flavoprotein spectrum either by reduction with dithionite and reoxidation under air, or by removal of the prosthetic group at pH 2 and reconstitution with FAD (not FMN). Besides FAD (4 mol/169 kDa), the enzyme contained 4 mol of a CoA ester which was similar but not identical to 5‐hydroxy‐2‐pentenoyl‐CoA. The reconstituted holoenzyme as well as the native green enzyme, but not the apoenzyme, catalysed the reversible dehydration of 5‐hydroxyvaleryl‐CoA to 4‐pentenoyl‐CoA in the absence of an external electron acceptor. In its presence (preferentially ferricenium ion), the green or yellow enzyme catalysed the formation of (E)‐5‐hydroxy‐2‐pentenoyl‐CoA and 2,4‐pentadienoyl‐CoA either from 4‐pentenoyl‐CoA or from 5‐hydroxyvaleryl‐CoA. The reversible hydration of 2,4‐pentadienoyl‐CoA to (E)‐5‐hydroxy‐2‐pentenoyl‐CoA was mediated by both enzymes as well as by the apoenzyme in the absence of FAD. Hydration of 4‐pentenoate in 2H2O yielded optically active 5‐hydroxy[2,4‐2H2]valerate by the combined action of 5‐hydroxyvalerate CoA‐transferase, the green dehydratase and catalytical amounts of acetyl‐CoA. The data show that the reversible hydration of the isolated double bond of 4‐pentenoyl‐CoA to 5‐hydroxyvaleryl‐CoA, which apparently violates the Markovnikov rule, is preceded by oxidation to 2,4‐pentadienoyl‐CoA. The latter compound, a vinyl analogue of 2‐enoyl‐CoA, is then easily hydrated to (E)‐5‐hydroxy‐2‐pentenoyl‐CoA and finally reduced to 5‐hydroxyvaleryl‐CoA.
Effects of mitochondrial swelling and calcium on phosphate‐activated glutaminase in pig renal mitochondria
Authors: KVAMME Elling , ROBERG Bjørg , TORGNER Ingeborg Aa.
Published: March 2005

The effects of mitochondrial swelling and calcium have been used to study the possible function of the glutamine transporter in regulating glutamine hydrolysis. Salt‐induced swelling of pig renal mitochondria and an iso‐osmotic mixed salt solution and swelling caused by reducing the osmolarity of the incubation medium, are accompanied by activation of glutamine hydrolysis. Regulation of the glutaminase activity by salt‐induced mitochondrial swelling is likely to have physiological importance, similar to the regulation of hepatic glutaminase by changing the matrix volume, that has been described by others.

0.1 – 1.0 mM calcium stimulates glutamine hydrolysis and the calcium activation curve follows Michaelis‐Menten kinetics. The calcium activation is reversible, it is unaffected by phosphate, high glutamine and mitochondrial calcium uptake, as well as by sonication and the activation is calmodulin independent. The calcium activation is additive to that of swelling.

Similar to calcium, hypo‐osmotic swelling mainly increases the apparent Vmax for glutamine, whereas the apparent Km is little changed, indicating that the effects are primarily on the phosphate‐activated glutaminase itself rather than on the glutamine transporter. Furthermore, calcium which activates glutamine hydrolysis, inhibits glutamine uptake into the mitochondria and so does alanine having no effect on glutamine hydrolysis. Therefore, it is indicative that glutamine transport is not rate limiting for glutamine hydrolysis.
Assignment of active‐site protons in the 1H‐NMR spectrum of reduced human Cu/Zn superoxide dismutase
Authors: BERTINI Ivano , CAPOZZI Francesco , LUCHINAT Claudio , PICCIOLI Mario , VIEZZOLI Maria Silvia
Published: March 2005

600‐MHz 1H‐NMR and nuclear Overhauser enhancement spectroscopy (NOESY) spectra in 2H2O and H2O, as well as truncated driven NOE difference spectra in H2O of reduced human Cu(II)2Zn(II)2 superoxide dismutase (Cu/Zn SOD) have been recorded and used to assign the active‐site proton signals. A derivative with histidines selectively deuteriated in the C2 position has been used for the detection of the HC2 histidine protons. 16 out of 17 observed signals of the 18 active‐site histidine ring protons have been assigned. The results are compared with previous proposals based on more limited data sets. The numerous cross peaks confirm that the structure in solution is essentially similar to the crystallographic data obtained on the oxidized form. Probably this holds also for His63 which in the reduced form is not bridging any more the two metal ions. The effects of azide binding on the exchangeable 1H‐NMR signals of the reduced protein are also reported.
Signal amplification in HL‐60 granulocytes
Authors: GIERSCHIK Peter , MOGHTADER Ruth , STRAUB Christoph , DIETERICH Klaus , JAKOBS Karl H.
Published: March 2005

Receptors for the chemotactic peptide fMet‐Leu‐Phe (fMet, N‐formylmethionine) are present in membranes of myeloid differentiated human leukemia (HL‐60) cells and stimulate phospholipase C via a pertussis‐toxin‐sensitive guanine‐nucleotide‐binding regulatory protein(s) [G‐protein(s)]. We have developed methods for the assessment of formyl‐peptide‐receptor‐stimulated binding of radiolabeled guanosine 5′‐[γ‐thio]triphosphate ([35S]GTP[S]) to native HL‐60 membranes. Agonist stimulation of [35S]GTP[S] association with the membrane was minimal ( 20%) when GTP[S] was the sole nucleotide present in the incubation medium. In contrast, receptor activation led to a marked (up to sixfold) stimulation of [35S]GTP[S] binding when GDP or GTP were present in high (> 100‐fold) excess of [35S]GTP[S]. The increase in [35S]GTP[S] binding caused by the chemotactic agonist was strictly dependent on the presence of Mg2+ and was significantly increased by Na+. Agonist‐independent binding of [35S]GTP[S] and the increase due to the chemotactic agonist were markedly attenuated by both pertussis and cholera toxin. Comparision of the number of chemotactic‐peptide‐sensitive [35S]GTP[S]‐binding sites to the number of chemotactic peptide receptors present in HL‐60 membranes provided direct evidence that a single formyl‐peptide receptor is capable of catalyzing the binding of [35S]GTP[S] to, and thus the activation of, multiple (up to 20) G‐proteins in native plasma membranes.
Proton nuclear magnetic resonance investigation of adrenodoxin
Authors: MIURA Shigetoshi , ICHIKAWA Yoshiyuki
Published: March 2005

Bovine, porcine and sheep adrenodoxin, and the trypsin‐resistant form of bovine adrenodoxin have been studied by one‐ and two‐dimensional 1H‐NMR spectroscopy. Assignment of the resonances for all the aromatic amino acids with resolved aromatic resonances have been made by correlating NMR spectra with the amino acid sequences from various species. Slowly exchanging amide protons and downfield shifted α‐protons of His10 and Phe11 suggest possible involvement in β‐sheet structure. The effects on the assigned resonances due to the specific spin‐lable with a nitroxide radical at Cys95 have been analyzed on a two‐dimensional 1H‐NMR spectrum. The present results provide evidence for a structural similarity with a model for the structure of adrenodoxin based on a sequence alignment with that of Spirulina platensis ferredoxin, for which X‐ray crystallographic data is available. ɛ‐Methyl groups of Met120 and Met122 have been assigned by comparing 1H‐NMR spectra of adrenodoxin with those of the trypsin‐resistant form of adrenodoxin which is specifically cleaved at Arg115. ɛ‐Methyl groups of Met120 and Met122 have an exceptionally long longitudinal relaxation time compared with those of valyl and leucyl methyl groups, suggesting that the COOH‐terminal peptide spanning over 13 amino acids rotates rather freely in the solvent.
Exonuclease activities associated with DNA polymerases α and β of the archaebacterium Halobacterium halobium
Authors: SOROKINE Irène , BEN‐MAHREZ Kamel , NAKAYAMA Masachi , KOHIYAMA Masamichi
Published: March 2005

α‐like and β‐like DNA polymerases have previously been isolated from a halophilic archaebacterium Halobacterium halobium. In this report, we show that the α‐like DNA polymerase has an associated 3′ to 5′‐exonuclease activity which is specific for single‐stranded DNA, sensitive to both aphidicolin and N‐ethylmaleimide and dependent on high salt concentrations like the polymerase activity. As this DNA polymerase has been shown to contain a primase activity, it may be considered as the equivalent to both eukaryotic DNA polymerases α and δ. As shown by glycerol‐gradient centrifugation and electrophoresis under denaturing conditions, the β‐like polymerase would appear to have a monomeric structure and comprise of a single 65‐kDa polypeptide. This DNA polymerase has both 3′ to 5′‐exonuclease and 5′ to 3′‐exonuclease activities which, contrary to polymerase activity, are inhibited by high salt concentrations.
Ethanol potentiates the stimulatory effects of phorbol ester, sphingosine and 4‐hydroxynonenal on the hydrolysis of phosphatidylethanolamine in NIH 3T3 cells
Authors: KISS Zoltan , CRILLY Karan , CHATTOPADHYAY Jyotiprakas
Published: March 2005

Ethanol and other alcohols have been shown to specifically stimulate phospholipase‐d‐mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Here, we further examined the possible mechanism of this ethanol action. Ethanol (10–300 mM) and the protein kinase C (PKC) activator 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) had synergistic stimulatory effects on the degradation of preformed [14C]PtdEtn when added in combination to [14C]ethanolamine‐labelled suspended NIH 3T3 cells 30 min after collection of cells by scraping. Scraping caused a transient increase, lasting for less than 30 min, in the cellular content of 1,2‐diacylglycerol, another PKC activator. Initially (0–50 min incubation), the main water‐soluble product of [14C]PtdEtn degradation in ethanol plus TPA‐treated cells was [14C]ethanolamine, while later (90 min) the main product of [14C]PtdEtn hydrolysis was [14C]ethanolamine phosphate in the presence of these agents. Ethanol also potentiated the specific stimulatory effects of sphingosine (through phospholipase D) and 4‐hydroxynonenal (not involving phospholipase D) on PtdEtn hydrolysis. The effects of these latter agents were unrelated to PKC activation. These data indicate that the observed potentiating effects of ethanol on PtdEtn hydrolysis do not involve direct regulation of PKC or phospholipase D activities.
Thioredoxin activation of phosphoribulokinase in a chloroplast multi‐enzyme complex
Authors: RAULT Magali , GONTERO Brigitte , RICARD Jacques
Published: March 2005

The activation process of spinach phosphoribulokinase by thioredoxin f has been studied with the enzyme in a free, isolated state, or integrated in a multi‐enzyme complex. The time periods required for enzyme activation are always smaller and the maximal enzyme velocities are always greater when chloroplast phosphoribulokinase is included in the multi‐enzyme complex than when it is in the isolated state. Comparative kinetic studies show that phosphoribulokinase extracted from the complex behaves exactly as in the isolated state. The reduced form of the kinase, whatever it has been included in the complex or isolated from the chloroplasts, are deactivated by oxidized thioredoxins. In the absence of thioredoxin f however, the reduced form of the isolated enzyme undergoes spontaneous oxidation whereas the reduced kinase included in the multi‐enzyme complex is stable. ‘Unspecific’ proteins such as bovine serum albumin do not provide any protection of the kinase against autooxidation, whereas ‘homologous’ specific proteins such as ribulose‐1,5‐bisphosphate carboxylase/oxygenase dramatically decrease the rate of this autooxidation process. These results therefore support the view that interactions between phosphoribulokinase and the other components of the multi‐enzyme complex play an important role in the modulation of the activity of this enzyme. The possible part of these interactions in the control of the Calvin cycle is discussed.
Isolation and characterization of recombinant human casein kinase II subunits α and β from bacteria
Authors: GRANKOWSKI Nikodem , BOLDYREFF Brigitte , ISSINGER Olaf‐Georg
Published: March 2005

cDNA encoding the casein kinase II (CKII) subunits α and β of human origin were expressed in Escherichia coli using expression vector pT7–7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII α subunit was purified by DEAE‐cellulose chromatography, followed by phosphocellulose and heparin‐agarose chromatography. The recombinant CKII β subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg α subunit and 5 mg β subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the α and β subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant α subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, α subunit kinase activity declined rapidly. Addition of the β subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 μM) was comparable to the mammalian enzyme (40–60 μM), whereas the α subunit alone had a Km of 240 μM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.
Binding of sugar phosphates, inositol phosphates and phosphorylated amino acids to actin
Authors: GAERTNER Andrea , MAYR Georg W. , WEGNER Albrecht
Published: March 2005

Binding of biological phosphate compounds to actin was investigated by the effect of these compounds on the critical concentration of the pointed ends of gelsolin‐capped actin filaments. According to this assay millimolar concentrations of glucose 6‐phosphate and the bisphosphorylated sugars fructose 1,6‐bisphosphate, fructose 2,6‐bisphosphate, glucose 1,6‐bisphosphate, sedoheptulose 1,7‐bisphosphate and 2,3‐bisphosphoglycerate were found to associate with actin. Glycerophosphoinositol phosphates bound to actin if they were present in millimolar concentrations, and if carbon atom 4 of the inositol ring was phosphorylated and carbon atom 5 was free of phosphate. Also phosphoserine and phosphotyrosine were found to interact with actin. Most of the actin‐binding compounds stabilized actin filaments by decreasing the critical concentration suggesting that these compounds had a higher affinity for the subunits along actin filaments than for actin monomers. However, 2,3‐bisphosphoglycerate and fructose 2,6‐bisphosphate increased the critical concentration probably because these sugar phosphates bound to actin monomers thereby inhibiting actin polymerization.
Repetitive sequences involved in the recombination leading to deletion of exon 5 of the low‐density‐lipoprotein receptor gene in a patient with familial hypercholesterolemia
Authors: RÜDIGER Niels S. , HANSEN Peter S. , JØRGENSEN Margrethe , FAERGEMAN Ole , BOLUND Lars , GREGERSEN Niels
Published: March 2005

Alu sequences in the low‐density‐lipoprotein (LDL) receptor gene are suspected of being of importance for the creation of gene defects leading to familial hypercholesterolemia (FH). One potential mechanism is that Alu sequences undergo homologous recombination, producing deletions or duplications of DNA segments on genomic DNA. In at least four cases (FH626, PO, JA and FH‐DK3), a deletion of exon 5 of the LDL receptor gene has been reported. Only one of these (FH626) have so far been characterized in detail by sequence analysis and shown to involve two of the Alu repeated sequences, which are present in introns 4 and 5. We here report the complete characterization of FH‐DK3 and show that the cross‐over break points involve sequences similar, but not at identical positions in the 5′ end, to those reported for FH626. The recombinations in both FH‐DK3 and FH626 are suggested to have occurred within a 22‐bp repeated sequence found in both junction alleles.
Resonant recognition model and protein topography
Authors: COSIC Irena , HODDER Anthony N. , AGUILAR Marie‐Isabel , HEARN Milton T. W.
Published: March 2005

This study describes the further extension of the resonant recognition model for the analysis and prediction of protein – protein and protein – DNA structure/function dependencies. The model is based on the significant correlation between spectra of numerical presentations of the amino acid or nucleotide sequences of proteins and their coded biological activity. According to this physico‐mathematical method, it is possible to define amino acids in the sequence which are predicted to be the most critical for protein function. Using sperm whale myoglobin, human hemoglobin and hen egg white lysozyme as model protein examples, sets of predicted amino acids, or so‐called ‘hot spots’, have been identified within the tertiary structure. It was found for each protein that the predicted ‘hot spots’, which are distributed along the primary sequence, are spatially grouped in a dome‐like arrangement over the active site. The identified amino acids did not correspond to the amino acid residues which are involved in the chemical reaction site of these proteins. It is thus proposed that the resonant recognition model helps to identify amino acid residues which are important for the creation of the molecular structure around the catalytic active site and also the associated physical field conditions required for biorecognition, docking of the specific substrate and full biological activity.
Ca2+‐dependent phospholipid and arachidonic acid binding by the placental annexins VI and IV
Authors: EDWARDS Helena C. , CRUMPTON Michael J.
Published: March 2005

Using an assay system in which phospholipids were immobilised on phenyl‐Sepharose, we examined the affinities of the placental annexins VI and IV for binding to specific phospholipids over a range of Ca2+ concentrations. Purified annexin VI showed half‐maximal binding to phosphatidylserine, phosphatidylethanol‐amine and phosphatidylinositol at Ca2+ concentrations of 0.6, 0.4 and 3.5 μM, respectively, compared to values of 4.5, 4.5 and 20 μM Ca2+, respectively for purified annexin IV. These values did not change significantly in the presence of other proteins from the family. Neither annexin VI or IV bound to phosphatidylinositol bisphosphate and phosphatidylcholine, even at millimolar concentrations of Ca2+. However, both proteins bound to arachidonic acid, oleic acid and palmitic acid in a Ca2+‐dependent manner, using the same assay system. The level of binding for both proteins was significantly increased when mixtures of phosphatidylcholine and arachidonic acid were examined. A dose‐dependent inhibition of phospholipase A2 by both annexins VI and IV, at millimolar concentrations of Ca2+ was observed when phosphatidylcholine liposomes were used as a substrate.

These results raise questions about the interpretation of experiments in which the release of arachidonic acid is used as a measure of lipase activity, and of the validity of the substrate‐depletion model for the inhibition of phospholipases by the annexins.
Isolation and structural characterization of novel neutral oligosaccharide‐alditols from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis
Authors: van KUIK J. Albert , de WAARD Pieter , VLIEGENTHART Johannes F. G. , KLEIN André , CARNOY Christophe , LAMBLIN Geneviève , ROUSSEL Philippe
Published: March 2005

Oligosaccharide‐alditols derived from the respiratory mucins of a patient suffering from bronchiectasis were separated by HPLC into 46 fractions. The structures of 11 oligosaccharides with GlcNAcβ(1→3)Galβ(1→4)‐GlcNAcβ(1→6)GalNAc‐ol in common have been established (preceding paper in this journal). In this second part the structures of 12 oligosaccharides were established 8 of which have not been described before. Of the 12 compounds, 6 possess the GlcNAcβ(1→3)[Galβ(1→4)GlcNAcβ(1→6)]Galβ(1→3)GalNAc‐ol element:
image

The other compounds, that could not be classified on basis of a common structural element, are:
image
Comparison of multiple forms of the high mobility group I proteins in rodent and human cells
Authors: GIANCOTTI Vincenzo , BANDIERA Antonella , BURATTI Emanuele , FUSCO Alfredo , MARZARI Roberto , COLES Brian , GOODWIN Graham H.
Published: March 2005

The class I of the high mobility group (HMG) proteins is formed by phosphoproteins which are associated with AT‐rich DNA sequences in the nucleus. Three HMGI proteins have previously been described in proliferating rodent cells (HMG Y, HMG I and HMGI‐C). All three proteins exhibit microheterogeneity. The microheterogeneity of mouse HMG Y has been investigated in detail and shown to be due to phosphorylation of the protein which is sensitive to alkaline‐phosphatase treatment. HMG I is similarly modified. Human cells have up to now only been found to contain HMG Y and HMG I. A search for the third protein, HMGI‐C, in human cells was carried out and the protein was found in a hepatoma cell line, but not in normal or transformed T‐cells. This HMGI‐C protein was found to be modified by phosphorylation, part of which was found to be phosphatase insensitive. An unexpected additional finding in this study was that human cells contain two HMG17 proteins which differ in their N‐terminal primary sequences.
Production and purification of recombinant human interleukin‐6 secreted by the yeast Saccharomyces cerevisiae
Authors: GUISEZ Yves , TISON Bernard , VANDEKERCKHOVE Joël , DEMOLDER Jan , BAUW Guy , HAEGEMAN Guy , FIERS Walter , CONTRERAS Roland
Published: March 2005

The coding region of the human interleukin‐6 (hIL6) gene was fused to the prepro secretion signal of the α‐mating factor gene in several yeast host strains. It was found that the KEX‐2 protease was unable to cleave the prepro‐Lys‐Arg‐Pro‐IL6 sequence, but that unspecific cleavage of the precursor protein had occurred. The prepro‐Lys‐Arg‐Ala‐Pro‐IL6 sequence, however, was correctly recognized and cleaved by the KEX‐2 protease, and IL6 was efficiently secreted into the culture medium. The N‐terminal Ala‐Pro peptide was removed during processing by wild‐type yeast strains, but was retained in a ste13 mutant. IL6 as well as the aberrant proteins were not glycosylated. The transformed cells could secrete up to 30 μg/ml IL6. The protein was purified from the medium to homogeneity by ion‐exchange chromatography and gel filtration, and had a specific activity of about 2 × 108 IU/mg in a proliferation assay.
Fluorescence study of the conformational properties of myoglobin structure
Authors: POSTNIKOVA Galina B. , KOMAROV Yuri E. , YUMAKOVA Elena M.
Published: March 2005

The pH‐dependent fluorescence of intact sperm whale apomyoglobin (apo‐Mb) containing two tryptophans at positions 7 and 14, and of apo‐Mb derivatives modified on Trp7 by 2‐hydroxy‐5‐nitrobenzyl bromide (Koshland reagent) and o‐nitrophenylsulphenyl chloride, has been studied. The fluorescence of apomyoglobins modified at His residues by iodoacetamide and bromoacetate, and at the N‐terminal α‐NH2 group by methylisothiocyanate, has also been investigated. The individual fluorescent properties of both tryptophans and their contributions to the total spectrum of apo‐Mb have been resolved within the pH range 2–12.5. The quantum yield of the ‘buried’ Trp14 (λmax at 326 nm) is shown to be twofold higher at pH >8.5 than that of the ‘exposed’ Trp7 (λmax at 333 nm). At pH 8.5–5.5 the fluorescence of Trp14 diminished approximately twofold due to quenching by the ionized His residue, most probably His119. The quenching is evidently dynamic because the fluorescence lifetime is shown to be linearly proportional to quantum yield in this pH range. The fluorescence of Trp7 practically does not change between pH 5.5 and 10.0 but increases 2.5–3‐fold in the pH range 5.5–4.3 while the contribution of Trp14 remains constant. The conformational changes at the N‐terminal and in the region adjacent to it, as well as in the whole apo‐Mb molecule in acidic, alkaline and neutral pH ranges, are considered. A relationship is revealed between conformational states of the heme crevice and the N‐terminal part of apo‐Mb.