bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced casein kinase II activity during mouse embryogenesis
Authors: SCHNEIDER Helge R. , REICHERT Gerd H. , ISSINGER Olaf‐Georg
Published: March 2005

Mouse embryos at various stages of development were used to study the relationship of protein kinase activities with normal embryogenesis. Casein kinase II (CKII) activity in developing mouse embryos shows a 3–4‐fold activity increase at day 12 of gestation. Together with the CKII activity, increased phosphorylation of a 110‐kDa protein is observed. Treatment of the embryo extracts with heparin, a highly specific inhibitor of CKII activity, results in a drastic reduction of the 110‐kDa protein phosphorylation indicating that the protein might be a CKII‐specific substrate. Rapidly proliferating mouse tumour cells also show an enhanced CKII activity. Here too, a 110‐kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP‐and cGMP‐dependent protein kinases) only show a basal level of enzyme activity with minor alterations throughout the different stages of embryogenesis investigated.
Partial purification of the maturation‐promoting factor MPF from unfertilized eggs of Xenopus laevis
Authors: NGUYEN‐GIA Pascal , BOMSEL Morgane , LABROUSSE Jean Pierre , GALLIEN Claude Louis , WEINTRAUB Hadassa
Published: March 2005

A 200‐fold purification of the maturation‐promoting factor or MPF from unfertilized eggs of Xenopus laevis is reported for the first time. Purification was achieved by three successive column chromatographies on hydroxyapatite, trisacryl blue and l‐arginine‐agarose. The presence of MPF was assessed by the usual maturation criteria after injections of test material into immature stage VI unstimulated X. laevis oocytes: the precocious appearance of the maturation spot (within 45–120 min), the germinal vesicle breakdown, the presence of the first polar body and the second metaphase spindle. Purification was monitored by the decrease of the minimal amount of protein injected in a constant volume (50 nl) required to induce 50% frequency of germinal vesicle breakdown. This amount decreased from 500 ng in the crude extract to 2.5 ng in the 200‐fold purified material. Analysis by SDS‐PAGE of the crude extract showed about 40 Coomassie‐blue‐stained polypeptides with molecular masses ranging from 300 kDa to 20 kDa, whereas in the 200‐fold purified MPF only 5 stained polypeptides were revealed, with molecular masses of 62, 53, 49, 39 and 37 kDa. In vitro phosphorylations for the detection of kinase activities for endogenous and exogenous substrates were monitored by analysis of autoradiograms of SDS‐PAGE, after treatment of fractions with [γ‐32P]ATP. Only inactive fractions eluted from columns ahead of MPF, and fractions containing MPF activity were tested. Phosphorylation of numerous stained polypeptides was demonstrated in the crude MPF extract and exogenous substrates such as phosvitin, casein and histone type II‐AS were also strongly phosphorylated. In the MPF fraction, purified on hydroxyapatite, a polypeptide of 53 kDa was more highly and specifically phosphorylated and the presence of kinase activities was observed for the above three exogenous substrates. In the 100‐fold and 200‐fold purified MPF, phosphorylation of endogenous substrates could not be shown and kinase activities for the above three substrates were drastically decreased as compared with the crude and purified MPF obtained after hydroxyapatite column chromatography. However, neither endogenous phosphorylation nor kinase activities with the above exogenous substrates could be shown in inactive fractions eluted ahead of MPF at the different purification steps. Some characteristics of the purified material are also described in this paper.
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.
Detection in situ of active polypeptides of mammalian and T4 DNA kinases after sodium dodecyl sulfate/polyacrylamide gel electrophoresis
Authors: OHMURA Yoshihisa , UCHIDA Tetsuo , TERAOKA Hirobumi , TSUKADA Kinji
Published: March 2005

DNA kinase activity of rat liver nuclei was detected in situ after electrophoresis in sodium dodecyl sulfate/polyacrylamide gel containing 5′‐hydroxyl nicked DNA as DNA substrate. After renaturation of polypeptides, the gel was incubated with [γ‐32P]ATP and Mg2+. An active polypeptide corresponding to Mr 61000 was observed as a radioactive band by autoradiography. The intensity of the band was proportional to the amount of the enzyme applied. The active band common to various tissues of rat was observed with the nuclear extracts, indicating that DNA kinase for rat tissues is composed of a single polypeptide of Mr 61000. In contrast, T4 polynucleotide kinase (Mr= 140000) showed an active polypeptide band corresponding to the subunit of Mr 33000.
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.
Further biochemical characterization of an Na+ pump inhibitor purified from human urine
Authors: CRABOS Maryse , GRICHOIS Marie‐Laure , GUICHENEY Pascale , WAINER Irving W. , CLOIX Jean‐François
Published: March 2005

An increase in endogenous Na+,K+‐ATPase inhibitor(s) with digitalis‐like properties has been reported in chronic renal insufficiency, in Na+‐dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis‐like properties. The urine‐derived inhibitor (endalin) inhibits Na+,K+‐ATPase activity and [3H]‐ouabain binding, and cross‐reacts with antidigoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+‐ATPase than on Mg2+‐ATPase and Ca2+‐ATPase. The mechanism of endalin action on highly purified Na+,K+‐ATPase was compared to that of ouabain and was similar in that (a) it reversibly inhibited Na+,K+‐ATPase activity; (b) it inhibited Na+,K+‐ATPase non‐competitively with ATP; (c) its inhibitory effect was facilitated by Na+; (d) K+ decreased its inhibitory effect on Na+,K+‐ATPase; (e) it competitively inhibited ouabain binding to the enzyme; (f) its binding was maximal in the presence of Mg2+ and Pi; (g) it decreased the Na+ pump activity in human erythrocytes; (h) it reduced serotonin uptake by human platelets; and (j) it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: (a) its inhibitory effect was not really specific for Na+,K+‐ATPase; (b) its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; (c) it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+‐ATPase, its Na+,K+‐ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.
Characterization of three isoenzymes of rat alcohol dehydrogenase
Authors: JULIÀ Pere , FARRÉS Jaume , PARÉS Xavier
Published: March 2005

Rat tissues contain three different isoenzymes of alcohol dehydrogenase (ADH) that we have named ADH‐1, ADH‐2 and ADH‐3. ADH‐1 is an anodic isoenzyme present in high amounts in the ocular tissues, stomach and lung. ADH‐2 is also anodic and has been found in all the rat organs examined. ADH‐3 is the group of cathodic ADH forms, mainly present in liver, that has been the subject of the majority of the previous studies on rat ADH. The three isoenzymes have been purified to homogeneity and characterized. All of them have similar physical characteristics: Mr 80 000, with two subunits of Mr 40 000; they contain four atoms of Zn per molecule, and prefer NAD+ as cofactor. Isoelectric points are, however, different: 5.1 for ADH‐1, 5.95–6.3 for ADH‐2 and 8.25–8.4 for ADH‐3. ADH‐3 exhibits a Km for ethanol of 1.4 mM, a broad substrate specificity and is strongly inhibited by pyrazole (Ki= 0.4 μM). ADH‐2 shows substrate specificity toward long‐chain alcohols and aldehydes, cannot be saturated by ethanol and is practically insensitive to pyrazole (Ki= 78.4 mM). ADH‐1 has intermediate properties, with a Km for ethanol of 340 mM, a broad substrate specificity and Ki for pyrazole of 0.56 mM. Rat ADH‐1, ADH‐2 and ADH‐3 exhibit many analogies with human ADH classes II, III and I respectively. The specific localization and kinetic properties of rat ADH isoenzymes suggest that ADH‐1 and ADH‐3 may act as metabolic barriers to external alcohols and aldehydes whereas ADH‐2 may have a function in the metabolism of the endogenous long‐chain alcohols and aldehydes.
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.
Soybean hydrophobic protein
Authors: ODANI Shoji , KOIDE Takehiko , ONO Teruo , SETO Yasuhiko , TANAKA Tatsuo
Published: March 2005

A 9000‐Mr protein isolated from a 60% ethanolic extract of soybean (Glycine max) seeds has been characterized and fully sequenced. The protein consists of 80 amino acid residues with four disulfide bonds. It contains a large number of hydrophobic residues and lacks methionine, phenylalanine, tryptophan, lysine and histidine residues. The protein readily crystallizes from water but is quite soluble in aqueous organic solvents like 95% 1‐propanol. It aggregates to form large molecules (above 80 kDa) under ordinary denaturing conditions, such as 6 M guanidine · HCl and 8 M urea. Sequence analysis showed that the amino‐terminal four‐fifths is extremely hydrophobic and most of the acidic residues exist as their amide forms, and only the carboxyl‐terminal short segment is rather hydrophilic. A computer search for homology detected an unexpected similarity of this protein to rat prolactin; however, its significance could not be assessed and this protein appears to represent a hitherto unknown protein family. Although no biochemical activity could be detected, the existence in relatively high abundance (approx. 200 mg from 1 kg seeds) of this novel protein may suggest its physiological significance in the plant.
Primary structure of the Streptomyces R61 extracellular DD‐peptidase
Authors: JORIS Bernard , JACQUES Philippe , FRÈRE Jean‐Marie , GHUYSEN Jean‐Marie , VAN BEEUMEN Jozef
Published: March 2005

In order to confirm the Streptomyces codon usage, the Streptomyces R61 DD‐peptidase was fragmented by (a) cyanogen bromide cleavage of the carboxymethylated protein, (b) trypsin digestion of the carboxymethylated protein and (c) trypsin digestion of the protein treated with β‐iodopenicillinate and endoxo‐δ4‐tetrahydrophthalic acid. The isolated peptides, which altogether represented more than 50% of the polypeptide chain, were sequenced. The data thus obtained were in excellent agreement with the primary structure of the protein as deduced from the nucleotide sequence of the cloned gene. Though a weak acylating agent, β‐iodopenicillanate reacted selectively with the active site of the DD‐peptidase and formed an adduct which mas much more stable than that formed with benzylpenicillin, thus facilitating the isolation and characterization of the active‐site peptide.
Purification and characterization of a serine hydroxymethyltransferase from an obligate methylotroph, Hyphomicrobium methylovorum GM2
Authors: MIYAZAKI Silvia Susana , TOKI Shin‐ichiro , IZUMI Yoshikazu , YAMADA Hideaki
Published: March 2005

A serine hydroxymethyltransferase was purified to complete homogeneity from a serine‐producing obligate methylotroph, Hyphomicrobium methylovorum GM2. The enzyme has a molecular mass of about 98 kDa and consists of two subunits of identical molecular mass. The holoenzyme exhibits absorption maxima at 280 nm. 340 nm and 415 nm in potassium phosphate buffer, pH 7.3, the last of which shifts with a change in pH (6.0 – 7.5) and contains 2 mol pyridoxal phosphate/mol enzyme. The holoenzyme is converted to the apoenzyme on incubation with phenylhydrazine and reconstituted on the addition of pyridoxal phosphate. The enzyme activity was inhibited on the addition of several sulfhydryl‐modifying reagents and then recovered with 2‐mercaptoethanol. One sulfhydryl group per subunit was found to be responsible for the activity. Isoelectric focusing showed that the enzyme has a pI of 5.6. The Km values for glycine, L‐serine and DL‐β‐phenylserine are 0.046 mM, 0.15 mM and 33 mM respectively.
Amino acid sequence of the serine active‐site region of the medium‐chain S‐acyl fatty acid synthetase thioester hydrolase from rat mammary gland
Authors: RANDHAWA Zafar I. , NAGGERT Jürgen , BLACHER Russell W. , SMITH Stuart
Published: March 2005

Medium‐chain S‐acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete monomeric enzyme of 29 kDa, regulates the product specificity of the de novo lipogenic systems in certain specialized mammalian and avian tissues, such as mammary and uropygial glands. The amino acid sequence of a 57‐residue region containing the active site of the rat mammary gland enzyme has been established by a combination of amino acid and cDNA sequencing. Thioesterase II was radiolabeled with the serine esterase inhibitor [1,3‐14C]diisopropylfluorophosphate and digested sequentially with cyanogen bromide, Staphylococcus aureus V8 protease and trypsin. A radiolabeled tryptic peptide was isolated and sequenced by automated Edman degradation and the location of the active‐site residue established. The amino acid sequence was confirmed by sequencing an overlapping, unlabeled peptide, obtained by V8 digestion of the whole enzyme, and by dideoxynucleotide sequencing of a thioesterase II cDNA clone isolated from a λgt11 expression library. The active center contains the motif Gly‐Xaa‐Ser‐Xaa‐Gly, characteristic of the serine esterase family of enzymes. A seven‐residue region around the essential serine of the rat mammary thioesterase II, Phe‐Gly‐Met‐Ser‐Phe‐Gly‐Ser, is completely homologous with a region of the mallard uropygial thioesterase, recently analyzed by cDNA sequencing, indicating that this is likely to be the active site of the avian enzyme. Overall homology between the mammalian and avian enzymes for the 57‐amino‐acid residue region is 47% and suggests that the two enzymes may share a common evolutionary origin.
Localization of the phalloidin and nucleotide‐binding sites on actin
Authors: BARDEN Julian A. , MIKI Masao , HAMBLY Brett D. , G. DOS REMEDIOS Cristobal
Published: March 2005

Phalloidin was found to block nucleotide exchange in F‐actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy‐transfer spectroscopy. They are separated by less than 1.0 nm. Added confirmation of the close proximity of phalloidin to nucleotide was obtained by extracting a small peptide‐ATP complex from an actin digest. The peptide comprises residues 114–118, which are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815–2818]. The results suggest that phalloidin has two major effects. It traps actin monomers in a conformation which appears to be distinct from G‐actin and it stabilizes the structure of F‐actin, an event accompanied by the trapping of ADP.
Migration of O‐acetyl groups in N,O‐acetylneuraminic acids
Authors: KAMERLING Johannis P. , SCHAUER Roland , SHUKLA Ashok K. , STOLL Sabine , VAN HALBEEK Herman , F. G. VLIEGENTHART Johannes
Published: March 2005

Highly purified N‐acetyl‐4‐O‐acetylneuraminic acid (Neu4,5Ac2), N‐acetyl‐7‐O‐acetylneuraminic acid (Neu5,7Ac2) and N‐acetyl‐7,9‐di‐O‐acetylneuraminic acid (Neu5,7,9Ac3) were used to study spontaneous migrations of acetyl groups between hydroxyl groups. The techniques applied involved thin‐layer chromatography, gas‐liquid chromatography/mass spectrometry, high‐performance liquid chromatography and 360‐MHz 1H‐NMR spectroscopy. It was found that at pH values at which no significant de‐O‐acetylation is observed: (a) Neu5,7Ac2 can easily be transformed into Neu5,9Ac2, (b) Neu5,7,9Ac3 yields an equilibrium of Neu5,7,9Ac3 and Neu5,8,9Ac3 in a molar ratio of approximately 1:1, and (c) Neu4,5Ac2 does not give rise to O‐acetyl migrations. The importance of these findings is discussed in terms of the biosynthesis of O‐acetylated sialic acids.
13C‐NMR, 1H‐NMR and gas‐chromatography mass‐spectrometry studies of the biosynthesis of 13C‐enriched l‐lysine by Brevibacterium flavum
Authors: INBAR Livia , LAPIDOT Aviva
Published: March 2005

The basic metabolic pathways of lysine biosynthesis in Brevibacterium flavum, a strain which excretes excessive amounts of l‐lysine, have been followed by using two 13C‐labeled precursors. 13C‐ and 1H‐NMR spectroscopies in conjunction with gas chromatography mass spectrometry (GC‐MS) have revealed the various metabolic pathways leading to l‐[13C]lysine. Discrete metabolic pathways give rise to distinct labeling patterns. l‐Lysine resulting from [1‐13C]glucose fermentation is relatively specifically labeled: l‐[3,5‐13C]lysine is the main product. Experimental and theoretical approaches based on the 13C‐enrichment values of intracellular glutamate, a major intermediate metabolite, allowed us to assess the relative contribution of the major metabolic pathways forming lysine. The labeling pattern of glutamate reflects the isotope distribution in 2‐oxoglutarate. When [2‐13C]acetate is used as the sole carbon source in the culture, the energy‐producing steps of the Krebs cycle are essential. The higher activity of the Krebs cycle, when endogenous carbohydrates are exhausted from the culture, is indicated by the increased 13C enrichment in C‐1 of lysine and reveal a high content of isotopomers of four, five and six 13C atoms in the lysine molecule, pointing out that the four‐carbon intermediates of the cycle are being derived from the glyoxylate shunt pathway. Such a phenomenon does not occur in glucose fermentation. GC‐MS analyses of 13C enrichments and isotopomer distributions in metabolites and end products are in good agreement with the predicted contribution of each metabolic pathway. This new methodological approach of combined NMR and GC‐MS has been demonstrated to be applicable to various other metabolic studies.
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.
Nitrile hydratase of Pseudomonas chlororaphis B23
Authors: NAGASAWA Toru , NANBA Hironori , RYUNO Koichiro , TAKEUCHI Koji , YAMADA Hideaki
Published: March 2005

Nitrile hydratase of Pseudomonas chlororaphis B23 was completely stabilized by the addition of 22 mM n‐butyric acid. The enzyme was purified from extracts of methacrylamide‐induced cells of P. chlororaphis B23 in eight steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular mass of about 100 kDa and consists of four subunits identical in molecular mass (approximately 25 kDa). The enzyme contained approximately 4 mol iron/mol enzyme. The concentrated solution of highly purified nitrile hydratase had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 720 nm. A loss of enzyme activity occurred in parallel with the disappearance of the absorption in the visible range under a variety of conditions. The enzyme catalyzed stoichiometrically the hydration of nitrile to amide, and no formation of acid and ammonia were detected. The enzyme was active toward various aliphatic nitriles, particularly, nitriles with 3—6 carbon atoms, e.g. propionitrile, n‐butyronitrile, acrylonitrile and cyclopropyl cyanide, served as the most suitable substrates.
High‐resolution solid‐state 13C‐NMR study of carbons C‐5 and C‐12 of the chromophore of bovine rhodopsin
Authors: MOLLEVANGER Leo C. P. J. , KENTGENS Arno P. M. , PARDOEN Johannes A. , COURTIN Jacques M. L. , VEEMAN Wibren S. , LUGTENBURG Johan , de GRIP Willem J.
Published: March 2005

Solid‐state 13C magic‐angle spinning NMR spectroscopy has been employed to study the conformation of the 11‐cis‐retinylidene Schiff base chromophore in bovine rhodopsin. Spectra were obtained from lyophilized samples of bovine rhodopsin selectively 13C‐labeled at position C‐5 or C‐12 of the retinyl moiety, and reconstituted in the fully saturated branched‐chain phospholipid diphytanoyl glycerophosphocholine. Comparison of the NMR parameters for carbon C‐5 presented in this paper with those published for retinyl Schiff base model compounds and bacteriorhodopsin by Harbison and coworkers [Harbison et al. (1985) Biochemistry 24, 6955–6962], indicate that in bovine rhodopsin the C‐6–C‐7 single bond has the unperturbed cis conformation. This is in contrast to the 6‐S‐trans conformation found in bacteriorhodopsin. The NMR parameters for bovine [12‐13C]rhodopsin present evidence for the presence of a negative charge interacting with the retinyl moiety near C‐12, in agreement with the model for the opsin shift presented by Honig and Nakanishi and coworkers [Kakitani et al. (1985) Photochem. Photobiol. 41, 471–479].
The glycoprotein Ib complex of human blood platelets
Authors: WICKI Andreas N. , CLEMETSON Kenneth J.
Published: March 2005

Human glycoprotein Ib (GPIb) is a major integral membrane protein on human blood platelets responsible for the initial attachment of platelets to the exposed vascular subendothelium. In this report we describe an isolation method for a ‘GPIb complex’ as well as for its individual components. A three‐step procedure involving Triton X‐114 phase‐partition, affinity chromatography on wheat germ agglutinin and ion‐exchange chromatography on DEAE‐Sephacel yielded milligram quantities of purified GPIb complex. The single components of the complex were further purified by gel filtration on AcA34 in 0.1% sodium dodecyl sulfate. As well as GPIb, the complex contains GP17, actin‐binding protein, actin and a series of unidentified proteins with different molecular masses. In contrast to GPIbα, which is very rich in O‐linked oligosaccharides, sugar analysis revealed that GPIbβ and GP17 seem to have only N‐linked chains of the lactosamine type. The C‐terminal α‐chain remnant, which probably spans the plasma membrane, was identified and isolated for the first time. Western blotting with polyclonal rabbit anti‐GPIb antibodies and silver‐staining of one‐ or two‐dimensional dodecyl sulfate/polyacryl‐amide gels revealed that it has an apparent molecular mass of 20 kDa and is linked to GPIbβ by a disulfide bridge close to the membrane. The thrombin‐binding site on GPIb is located near the N‐terminus on a 40‐kDa fragment of GPIbα. A disulfide bridge in the N‐terminal region is not essential for thrombin binding to GPIb.
Ab initio quantum mechanical calculations of the variation of the 1H and 13C nuclear magnetic shielding constants in proline as a function of the angle ψ
Authors: GIESSNER‐PRETTRE Claude , CUNG Manh Thong , MARRAUD Michel
Published: March 2005

The variation of the nuclear magnetic shielding constant of the different protons and carbons of trans HCO‐l‐Pro‐NH2 with the value of the angle ϕ is calculated by a non‐empirical method for three conformations of the proline ring. The results concerning the CH protons show that the chemical shift of the α, β and γendo hydrogens can vary by more than 1 ppm when Ψ goes from −30° to 180°. The theoretical variation of the chemical shift difference between α and γ or β and γ carbons is found to be sensitive to the puckering of the proline ring. For the second of these differences the theoretical results are in agreement with Siemion's relation only for a limited range of molecular conformations. Additional calculations show that the variations of the proton shifts with the value of Ψ are due to the magnetic anisotropy of the proline carbonyl group and to the polarization of the CH bonds by the multipolar charge distribution carried by this carbonyl. The results are discussed in relation to experiment and the possibility of using 1H and 13C chemical shifts for the determination of the value of the torsion angle about the CαC′ bond.
Purification and characterisation of ribonuclease activities that interact with the cytoplasmic inhibitor protein of rat tissues
Authors: BROCKDORFF Neil A. , KNOWLER John T.
Published: March 2005

Evidence is presented that a number of different ribonuclease activities interact with the cytoplasmic ribonuclease inhibitor of rat and that these enzymes vary in their distribution in different tissues. Two ribonuclease activities were purified from rat liver and three from rat uterus. They were characterised with respect to their activity, substrate preference and pH optima.
Kinetics of the oxidation of ascorbic acid, ferrocyanide and p‐phenolsulfonic acid by chloroperoxidase compounds I and II
Authors: LAMBEIR Anne‐Marie , DUNFORD H. Brian , PICKARD Michael A.
Published: March 2005

For the first time elementary reactions involving chloroperoxidase compounds I and II have been investigated. A multi‐mixing stopped‐flow apparatus was used to study the kinetics of the reactions of compounds I and II with ascorbic acid, ferrocyanide and p‐phenolsulfonic acid. The second‐order rate constants of the reactions of both compounds with all three substrates were determined between pH 3 and pH 7. In all cases the rate constants decrease with increasing pH. The reactions of p‐phenolsulfonic acid are influenced by a catalytically important group on both compounds I and II with a pKa of 3.7 ± 0.2. With ascorbic acid and ferrocyanide as substrates, a decrease in rate was observed upon ionization of the substrate. Comparisons with horseradish peroxidase show that chloroperoxidase is a much less efficient peroxidatic enzyme. The kinetic data were used to calculate the percentage composition of the mixture of chloroperoxidase species which contribute to the spectra measured during the turnover with ascorbate as substrate.
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.
Differential expression of heat‐shock proteins and spontaneous synthesis of HSP70 during the life cycle of Blastocladiella emersonii
Authors: BONATO M. Christina M. , SILVA Aline M. , GOMES Suely L. , MAIA José Carlos C. , JULIANI Maria Helena
Published: March 2005

The heat‐shock response in Blastocladiella emersonii is dependent on the developmental stage. Cells exposed to elevated temperatures at different stages of the life cycle (sporulation, germination or growth) show a differential synthesis of heat‐shock proteins (hsps). Of a total of 22 polypeptides induced, particular subsets of hsps appear in each phase, demonstrating a non‐coordinate heat‐shock gene expression. In contrast, heat‐shock‐related proteins (hsp76, hsp70, hsp39a) are spontaneously expressed at a high level during sporulation. By the criteria of two‐dimensional gel electrophoresis and partial proteolysis mapping, the 70 000‐Da protein, whose synthesis is induced spontaneously during sporulation, is indistinguishable from the heat‐inducible hsp70. The techniques of in vitro translation, and Northern analysis using a Drosophila hsp70 probe, demonstrated that enhanced synthesis of hsp70, which occurs during heat‐shock treatment and spontaneously during sporulation, is associated with an accumulation of hsp70 mRNA. These observations suggest that hsp70 gene expression is induced during sporulation.
The A and B fragments of normal type I procollagen have a similar thermal stability to proteinase digestion but are selectively destabilized by structural mutations
Authors: CONSTANTINOU Constantinos D. , VOGEL Bruce E. , JEFFREY John J. , PROCKOP Darwin J.
Published: March 2005

Previous studies demonstrated that the thermal stability of the procollagen triple helix can be assayed by digesting the protein for short periods with high concentrations of trypsin and chymotrypsin. Here we cleaved human type I procollagen or collagen with vertebrate collagenase to generate A fragments from the three‐quarter amino termini and B fragments from the one‐quarter carboxy termini of the molecules. The thermal stabilities of the fragments were then assayed by rapid trypsin/chymotrypsin digestion. Both fragments were resistant up to 36°C and completely degraded between 37°C and 39°C. In subsequent experiments the same assay was carried out with type I procollagens synthesized by fibroblasts from two patients with lethal variants of osteogenesis imperfecta. With one, the A fragments were selectively destabilized, an observation consistent with previous data indicating that the mutation in the patient produced a deletion of 84 amino acids from the middle of the α1(I) chain. With procollagen synthesized by fibroblasts from the second patient the B fragments were selectively destabilized, an observation consistent with preliminary data indicating a mutation that alters the primary structure of the carboxy‐terminal region of the α1(I) chain. Therefore, the procedures described here present a simple and direct method for locating mutations that destabilize the collagen triple helix.
Studies on the stability of the higher‐order structure of rat liver chromatin containing high‐mobility‐group proteins
Authors: JOSE Matilde , PUIGDOMÈNECH Pere , PALAU Jaume
Published: March 2005

The stability of the higher‐order structure of chromatin containing high‐mobility‐group (HMG) proteins has been studied in rat liver nuclei by mild micrococcal nuclease digestion at low temperature and fractionation by sucrose gradient centrifugation. Nuclei preparation and digestion, chromatin solubilization and analysis have been carried out in two ionic conditions, 140 mM and 40 mM monovalent cation concentration, avoiding drastic changes in ionic conditions and temperature during preparation and analysis. During the time course of digestion at 140 mM ionic strength a material stable at 80 S appears, whose DNA is cleaved at values around 12 nucleosomes. The distribution of HMG proteins in different chromatin fractions was analyzed by immunodot using antibodies elicited against proteins HMG‐1, HMG‐2, and HMG‐14 and 17. It appears that these proteins have a distribution distinctly different from the bulk of chromatin. They are never found in the chromatin fragments that keep their internucleosomal interactions, indicating that these proteins tend to accumulate in points where the chromatin has a less stable structure.
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.
Kinetics of epidermal growth factor binding and processing in isolated intact rat hepatocytes
Authors: GLADHAUG Ivar P. , CHRISTOFFERSEN Thoralf
Published: March 2005

The kinetics of binding and processing of epidermal growth factor (EGF) was studied in freshly isolated rat hepatocytes. After isolation the hepatocytes had a nonhomogeneous population of surface EGF receptors consisting of approximately 9000 high‐affinity sites (Kd 21 pM) and 165000 low‐affinity sites (Kd 0.62 nM). Incubation at 37°C (45 min) increased the number of surface receptors per cell to about 260000. This increase was selective for the low‐affinity receptors and was cycloheximide‐sensitive. During 5 h of incubation at 37°C the hepatocytes internalized 6–7‐times more EGF molecules than the number of cell surface receptors, based on clearance measurements. The uptake was unaffected by cycloheximide. Concomitant estimation, using acid/salt elution, of surface‐bound EGF and internalized EGF showed that the number of internalized EGF molecules exceeded the decrease in surface‐binding 6 times. The ratio between internalized EGF and the decrease in surface binding was temperature‐dependent, being reduced to a one‐to‐one stoichiometry at 10°C. After down‐regulation (∼ 75%) induced by 5 nM unlabeled EGF the surface EGF receptors did not recover during subsequent incubation (2 h) at 37°C. However, the remaining surface receptors internalized EGF in ninefold excess of their number. The large discrepancy between internalization capacity and cell surface binding capacity was also found in the presence of cycloheximide. The results support the idea that internalized EGF receptors are partly replaced by externalization of preformed intracellular receptors during EGF uptake in isolated hepatocytes, involving recycling of a small population of EGF receptors and/or recruitment of unexposed, pre‐existing receptors.