bluuebunny/crossref_metadata_2025_split huggingface dataset:
Primary structure of the chromosomal protein HMb from the archaebacteria Methanosarcina barkeri
Authors: LAINE Bernard , CHARTIER François , IMBERT Marlène , LEWIS Randy , SAUTIERE Pierre 
Published: March 2005

The amino acid sequence of the protein HMb, a protein of 93 residues (Mr 10757) which represents the major acid‐soluble component of the Methanosarcina barkeri nucleoprotein complex, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid, arginine and methionine residues.The protein HMb is mainly characterized by a high amount of charged residues (15% of acidic residues and 26.8% of basic residues) which are distributed all along the polypeptide chain. The amino acid sequence of the protein HMb is not homologous to any eubacterial, archaebacterial or eukaryotic chromosomal proteins known up to now.
Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins
Authors: KITAGAKI‐OGAWA Haruko , MATSUMOTO Isamu , SENO Nobuko , TAKAHASHI Noriko , ENDO Satoshi , ARATA Yoji 
Published: March 2005

Lectins were isolated from fruits and leaves of Clerodendron trichotomum by affinity chromatograpy on lactamyl‐Sepharose. The purified lectins (C. trichotomum agglutinin: CTA) were homogeneous on SDS/polyacrylamide gel electrophoresis, and the carbohydrate moiety was characterized by physicochemical and immuno‐chemical methods.The asparagine‐linked oligosaccharides were released by treatment with N‐oligosaccharide glycopeptidase (almond, EC 3.5.1.52) of peptic glycopeptides obtained from fruit CTA, and separated by gel filtration and thin‐layer chromatography. The structure of the predominant oligosaccharide was determined as Xylβ1 → 2 (Manα1 → 6)(Man α1 → 3)Manβ1 → 4GlcNAcβ1 → 4(Fucα1 → 3)GlcNAc by high‐performance liquid chroma‐tography, sugar analysis and 1H‐NMR spectroscopy.The reactivity of the carbohydrate moiety of CTA toward various lectins was studied. Fruit and leaf CTAs were applied to polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and detected with horseradish‐peroxidase‐conjugated lectins. Concanavalin A, lentil lectin, pea lectin, Vicia faba lectin and Ulex europeus agglutinin I, but not wheat germ lectin, bound to fruit CTA. The results indicate new binding properties of these plant lectins: (a) a β‐xylosyl residue substituted at C‐2 of the β‐mannosyl residue of N‐linked oligosaccharide does not affect the binding with mannose‐specific lectins, (b) lentil, pea and Vicia faba lectins can bind to N‐linked oligosaccharides containing an α‐l‐fucosyl residue attached to C‐3 of the asparagine‐linked N‐acetyl‐D‐glucosamine residue, and (c) Ulex europeus agglutinin I can bind to the (α1 → 3)‐linked fucose residue of the N‐linked oligosaccharide.
Non‐covalent interactions between Fab and Fc regions in immunoglobulin G molecules
Authors: KILÁR Ferenc , ZÁVODSZKY Péter 
Published: March 2005

To reveal non‐covalent interactions between the Fab and Fc regions of IgG molecules the average conformational free‐energy change (), associated with reversible micro‐unfoldings, was measured by hydrogen–deuterium exchange for the Fab and Fc fragments and the complete molecule. Human monoclonal IgG1 and pooled IgG samples were used in these experiments.Hydrogen–deuterium exchange data were summarized and compared in the form of exchange relaxation spectra. The experimentally observed relaxation spectrum of intact IgG could not be deduced by weighted summation of spectra measured for Fab and Fc fragments. A comparison of the measured and calculated data revealed a 5‐kJ/mol increase in the conformational free energy upon splitting the IgG molecule into two Fab and Fc pieces, i.e. an increase of conformational mobility occurred. This change can be explained either by related fluctuation patterns of the Fab and Fc pieces in the intact molecule or by a shielding effect on the contact surfaces. Both interpretations suppose non‐convalent interactions between Fab and Fc that can be a means of information transduction between recognition and effector sites. The pH dependence of the hydrogen–deuterium exchange also indicates interactions between the Fab and Fc regions. A shift in the relaxation spectra of the Fab fragment was observed between pH 8.2 and 7.3 revealing destabilization of the structure at lower pH. This effect is absent in the intact molecule, reflecting interactions that stabilize the Fab structure. Comparison of the relaxation spectra of Fab and Fc shows a difference of about 10 kJ/mol in the microstability of these fragments: the Fab part possesses more conformational flexibility (i.e. its microstability is smaller) than the Fc part.
Purification and partial characterization of lysosomal neuraminidase from human placenta
Authors: VERHEIJEN Frans W. , PALMERI Silvia , GALJAARD Hans 
Published: March 2005

Lysosomal neuraminidase and β‐galactosidase are present in a complex together with a 32‐kDa protective protein. This complex has been purified and the different components have been dissociated using potassium isothiocyanate (KSCN) treatment. β‐Galactosidase remains catalytically active, but neuraminidase loses its activity upon dissociation. The inactive dissociated neuraminidase was purified by removing the remaining non‐dissociated β‐galactosidase/protective protein complex using β‐galactosidase‐specific affinity chromatography. The dissociated neuraminidase material shows two major polypeptides on SDS‐PAGE with an apparent molecular mass of 76 kDa and 66 kDa. Subsequently the 32‐kDa protective protein was dissociated from the β‐galactosidase/protective protein complex, and purified.Antibodies raised against the dissociated inactive neuraminidase preparation specifically immunoprecipitate the active neuraminidase present in the complex with β‐galactosidase and protective protein.By immunoblotting evidence is provided that the 76‐kDa protein is a subunit of neuraminidase which, in association with the 32‐kDa protective protein, is essential for neuraminidase activity.
The role of N‐glycosylation for the plasma clearance of rat liver secretory glycoproteins
Authors: GROSS Volker , STEUBE Klaus , TRAN‐THI Thuy‐Anh , HÄUSSINGER Dieter , LEGLER Günter , DECKER Karl , HEINRICH Peter C. , GEROK Wolfgang 
Published: March 2005

The clearance of total rat liver secretory glycoproteins and of α1‐acid glycoprotein carrying no or different types of oligosaccharide side chains was studied in vivo and in the isolated perfused rat liver. In order to obtain unglycosylated or differently glycosylated forms of secreted glycoproteins, rat hepatocyte primary cultures were incubated with various inhibitors of N‐glycosylation. Tunicamycin was used for the synthsis of unglycosylated (glyco)proteins, the mannosidase I inhibitor 1‐deoxymannojirimycin for the syntesis of high‐mannose type and the mannosidase II inhibitor swainsonine for the synthesis of hybrid‐type glycoproteins. Glycoproteins carrying carbohydrate side chains of the complex type were synthesized by control hepatocytes. In vivo and in the perfused rat liver, high‐mannose‐type glycoproteins were cleared at the highest rate, followed by unglycosylated and hybrid‐type glycoproteins. The lowest clearance rate was found for the glycoproteins with carbohydrate side chains of the complex type. For the highly glycosylated α1‐acid glycoprotein the differences in clearance rates were more pronounced. The following plasma half‐lives were determined in vivo: complex type, 100 min; hybrid type, 15 min; unglycosylated form, 5 min; and high‐mannose type less than 1 min. In the recirculating perfused liver 28% of complex‐type α1‐acid glycoprotein, 40% of hybrid type, 47% of unglycosylated and 93% of high‐mannose‐type α1‐acid glycoprotein were removed from the perfusate within 2 h. It is concluded that N‐glycosylation and processing to complex‐type oligosaccharides seems to be of great importance for the circulatory life time of plasma glycoproteins.
Chemical modification studies of beef‐heart mitochondrial b‐c1 complex
Authors: LORUSSO Michele , GATTI Domenico , MARZO Marcella , BOFFOLI Domenico , COCCO Tiziana , PAPA Sergio 
Published: March 2005

The effect of the histidine‐modifier ethoxyformic anhydride (EFA) on the enzymatic properties of the mitochondrial b‐c1 complex (ubiquinol–cytochrome c reductase) has been investigated. Chemical modification by EFA inhibited to the same extent the reductase and the proton translocating activity of the complex. In particular EFA modification of the complex resulted in: (a) strong inhibition of the antimycin‐insensitive reduction of b cytochromes; (b) inhibition of the antimycin‐promoted oxidant‐induced reduction of b cytochromes and (c) inhibition of oxidation of pre‐reduced b cytochromes.Analysis of the absorbance at 238 nm, indicative of N‐(ethoxyformyl)histidine derivative, of the various polypeptide subunits separated by high‐pressure liquid chromatography procedure, showed that EFA modified residues in core proteins and in the low‐molecular‐mass proteins. Both the inhibition of the redox and the protonmotive activity of the complex and the absorbance increase at 238 nm of the core protein fraction were readily reversed by hydroxylamine, indicating that modification of histidine residue(s) in core protein(s) is critical for the activity of the complex.This was supported by the finding that modification of the reductase with EFA prevented binding of fluoresceinisothiocyanate to histidine residue(s) in core protein II. EFA modification of the reductase was without effect on the binding of N‐(7‐dimethylamino‐4‐methylcoumarinil)maleimide to the various polypeptides of the complex except for the binding to the Fe‐S protein which was greatly potentiated. Thus primary chemical modification of histidine residue(s) in core protein (II) appears to cause, in turn, a conformational change in the Rieske Fe‐S protein.
Juvenile‐hormone‐binding protein from the hemolymph of Galleria mellonella (L)
Authors: OżYHAR Andrzej , KOCHMAN Marian 
Published: March 2005

A juvenile‐hormone‐binding protein (JHBP) has been isolated from Galleria mellonella hemolymph by gel filtration, phosphocellulose chromatography, and by chromatofocusing. The isolated protein is homogenous as judged by column chromatography and gel electrophoresis in the presence and absence of denaturing agent. It has a relative molecular mass of 32000, Stokes radius 2.4 nm, sedimentation coefficient of 2.3 S, molar absorption coefficient at 280 nm ɛ= 2.34 × 104 M‐1 cm‐1, and is composed of a single polypeptide chain.Chromatofocusing analysis (pI 8.6) and isoelectric focusing (pI 8.1) indicate that the JHBP is an alkaline protein. Its amino acid composition and fluorescence absorption spectra indicate that the protein does not contain tryptophan residues.The protein exhibits one class of binding sites for juvenile hormone (JH), 0.8 per molecule, with the following dissociation constants: JH I, 8.5 × 10‐8 M; JH II, 7.2 × 10‐8 M; JH III, 47 × 10‐8 M. The JHBP binds (10R, 11S)‐JH II enantiomer with 2.3‐times higher affinity then (10S, 11R)‐JH II enantiomer. The pH optimum of binding is 7.0.
Two new vitamin B‐12 factors from sewage sludge containing 2‐methylsulfinyladenine or 2‐methylsulfonyladenine as base component
Authors: RENZ Paul , BLICKLE Sylvia , FRIEDRICH Wilhelm 
Published: March 2005

Two hitherto unknown vitamin B‐12 factors were isolated from sewage sludge. They were degraded with cerous hydroxide to cobinamide and the corresponding nucleoside. The nucleosides were further split with dilute hydrochloric acid to the bases and d‐ribose. The structure of the two bases was found to be 2‐methylsulfinyladenine and 2‐methylsulfonyladenine. This was revealed by mass, infrared and nuclear magnetic resonance spectroscopy, and by comparison with the synthetic compounds.On addition of the synthetic bases to fermentations with Propionibacterium acidi‐propionici the vitamin B‐12 factors containing the corresponding base were formed. They were identical with the 2‐methylsulfinyl‐adenylcobamide and 2‐methylsulfonyladenylcobamide originally isolated from sewage sludge.
Perivascular nerve stimulation and phenylephrine responses in rat liver
Authors: HÄUSSINGER Dieter , STEHLE Thomas , GEROK Wolfgang , SIES Helmut 
Published: March 2005

Electrical stimulation of perivascular nerves (20 Hz/2 ms/20 V) in perfused rat liver led to a transient increase of 14CO2 production from [1‐14C]glutamate, glutathione and thiol efflux, an increase in the lactate/pyruvate and the 3‐hydroxybutyrate/acetoacetate ratio, glucose release and of portal pressure.
These metabolic effects were accompanied by a Ca2+ release from the liver within the initial 2 min, being followed by Ca2+ reuptake, which lasted about 3 min. The initial Ca2+ release was 67 nmol/g liver and was smaller than that observed after phenylephrine (5 μM) addition (156 nmol/g liver). Hepatic Ca2+ release following nerve stimulation or phenylephrine was not significantly affected when the hemodynamic changes were largely prevented by sodium nitroprusside (10 μM). Although the amounts of Ca2+ released were different, the glycogenolytic responses, but not the other metabolic effects, were quantitatively similar with nerve stimulation and phenylephrine.
Within the first 3 min of nerve stimulation there was a K+ uptake by the liver being followed by a K+ release over the next 5–6 min and a subsequent slow K+ uptake phase. These changes resembled those observed with phenylephrine. Phentolamine, an α‐adrenergic antagonist, abolished the Ca2+ and K+ movements following nerve stimulation as well as glucose release and the hemodynamic changes. During continuous infusion of phenylephrine, nerve stimulation led still to an increase of portal pressure; however, the effects of nerve stimulation on Ca2+ and K+ fluxes and glucose release were largely suppressed.
It is concluded that the metabolic effects of electrical nerve stimulation are mediated by a redistribution of cellular Ca2+ following α‐receptor activation. Nerve stimulation involves Ca2+ and K+ fluxes across the plasma membrane. The metabolic effects are qualitatively similar to those induced by phenylephrine. The quantitative difference between nerve stimulation and phenylephrine is explained by a differential subacinar response, with fewer cells being reached by nerve stimulation than cells containing α‐receptors. The hemodynamic changes of nerve stimulation point to the existence of sphincters near the inflow of the sinusoidal bed.
Inhibitors of protein kinase C block the α1‐adrenergic refractoriness induced by phorbol 12‐myristate 13‐acetate, vasopressin and angiotensin II
Authors: GARCÍA‐SÁINZ J. Adolfo , HERNÁNDEZ‐SOTOMAYOR S. M. Teresa 
Published: March 2005

Vasopressin and angiotensin II inhibited in a dose‐dependent fashion the stimulation of ureagenesis unduced by α1‐adrenergic activation in hepatocytes incubated in medium without calcium and containing 25 μM EGTA. Vasopressin was more potent than angiotensin II. The effect of different inhibitors of protein kinase C on the α1‐adrenergic blockade induced by the vasopressor peptides was tested. It was observed that N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalene sulfonamide (W‐7), 4‐aminoethyl‐1‐[2,3‐bis(n‐decloxyl)‐n‐propyl]‐4‐phenylpiperadine dihydrochloride (CP‐46,665‐1); 8‐(N,N‐diethylamino)octyl‐3,4,5‐trimethoxybenzoate (TMB‐8), polymyxin B and 1‐(5‐isoquinolynsulfonyl)‐2‐methylpiperazine (H‐7) block this effect of the vasopressor peptides in a dose‐dependent fashion.The active phorbol ester, phorbol 12‐myristate 13‐acetate (PMA), also inhibited the α1‐adrenergic stimulation of ureagenesis in these cells. The inhibitors of protein kinase also blocked the effect of phorbol esters but a preincubation with the inhibitors before the addition of PMA was required. α1‐Adrenergic activation of phosphatidylinositol labeling was also abolished by PMA; the inhibitors of protein kinase partially blocked this effect of PMA.In summary, our data indicate that inhibitors of protein kinase C can block the α1‐adrenergic refractoriness induced by active phorbol esters, vasopressin and angiotensin II. The data are consistent with an important role of protein kinase C in modulating the α1‐adrenergic responsiveness of hepatocytes.
Purification and characterization of hyoscyamine 6β‐hydroxylase from root cultures of Hyoscyamus niger L.
Authors: HASHIMOTO Takashi , YAMADA Yasuyuki 
Published: March 2005

Hyoscyamine 6β‐hydroxylase, a 2‐oxoglutarate‐dependent dioxygenase that catalyzes the hydroxylation of l‐hyoscyamine to 6β‐hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. & Yamada, Y. (1986) Plant Physiol. 81, 619–625] was purified 310‐fold from root cultures of Hyoscyamus niger L. The enzyme has an average Mr of 41000 as determined by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8. l‐Hyoscyamine and 2‐oxoglutarate are required for the enzyme activity, with respective Km values of 35 μM and 43 μM. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2‐Oxoglutarate was not replaced by any of ten other oxo acids tested, nor was Fe2+ by nine other divalent cations tested.The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic acids, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o‐dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4‐dicarboxylate and 3,4‐dihydroxybenzoate are competitive inhibitors with respect to 2‐oxoglutarate with the respective Ki values of 9 μM and 90 μM.Several alkaloids with structures similar to l‐hyoscyamine were hydroxylated by the enzyme at the C‐6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7‐dehydrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 μM). This epoxidation reaction required the same co‐factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6β‐hydroxylase.
Sterol biosynthesis via cycloartenol and other biochemical features related to photosynthetic phyla in the amoebae Naegleria lovaniensis and Naegleria gruberi
Authors: RAEDERSTORFF Daniel , ROHMER Michel 
Published: March 2005

The sterols and sterol precursors of two amoebae of the genus Naegleria, Naegleria lovaniensis and Naegleria gruberi were investigated. Cycloartenol, the sterol precursor in photosynthetic organisms, is present in both amoebae. In N. lovaniensis, it is accompanied by lanosterol and parkeol, as well as by the 24, 25‐dihydro derivatives of these triterpenes. One of the most striking features of these amoebae is the accumulation of 4α‐Methylsterols which are present in similar amounts as those of 4,4‐desmethylsterols (3–5 mg/g, dry weight). 4α‐Methylergosta‐7,22‐dienol was identified as a new compound. Ergosterol was the major 4,4‐desmethylsterol, accompanied by small amounts of C27 and other C28 sterols.Treatment of N. lovaniensis with fenpropimorph modified the sterol pattern of this amoeba and inhibited its growth. This fungicide, known to inhibit steps of sterol biosynthesis in fungi and plants, induced the disappearance of 4α‐methyl‐Δ7‐sterols and the appearance of the unusual Δ6,8,22‐ergostatrienol as in A. polyphaga. These results might be explained by a partial inhibition of the Δ8→Δ7 isomerase, the small amounts of Δ7‐sterols formed being converted into ergosterol which is still present in fenpropimorph‐exposed cells. De novo sterol biosynthesis in N. lovaniensis was shown by incorporation of [1‐14C]acetate into sterols and sterol precursors, especially cycloartenol. Lanosterol and parkeol were not significantly labelled. Furthermore, [3‐3H]squalene epoxide was efficiently cyclized by a cell‐free system of this amoeba into cycloartenol, and again no significant radioactivity was detected in lanosterol and parkeol. This shows that cycloartenol, the sterol precursor in plants and algae, is also the sterol precursor in Naegleria species, and that these amoebae, like A. polyphaga, are related by some biosynthetic pathways to photosynthetic phyla. Lanosterol, the sterol precursor in non‐photosynthetic phyla (animal and fungi) and parkeol are more likely dead‐ends of this biosynthetic pathway.The peculiar phylogenetic position of these protozoa was further emphasized by the action of indole acetic acid and other auxine‐like compounds on their growth. Indeed amoebic growth was enhanced in the presence of these higher plant growth hormones. The differences in the sterol composition of the protozoa we have hitherto examined is related to their sensitivity toward polyene macrolide antibiotics. The growth of A. polyphaga containing mainly the C29 stigmasta‐5,7,22‐trienol was not affected by nystatin, and only slightly by amphotericin. Both antibiotics inhibited however the growth of the ergosterol‐containing amoeba N. lovaniensis and trypanosome Crithidia fasciculata.
Application of a reference convolution method to tryptophan fluorescence in proteins
Authors: VOS Kees , van HOEK Arie , VISSER Antonie J. W. G. 
Published: March 2005

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p‐terphenyl, p‐bis[2‐(5‐phenyloxazolyl)]benzene and N‐acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p‐terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p‐terphenyl (20°C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile‐up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile‐up effects play a role at this frequency.The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple‐exponential fluorescence decay. The anisotropy decay of albumin was found to be mono‐exponential with a rotational correlation time of 26 ns (20°C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for λex= 295 nm and 36 ns for λex= 300 nm at 20°C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple‐exponential fluorescence decay. A single‐exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.