bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and properties of the mercuric‐ion‐binding protein MerP
Authors: SAHLMAN Lena , JONSSON Bengt‐Harald
Published: March 2005

The gene merP, coding for a mercuric‐ion‐binding periplasmic protein (P protein), was cloned into the expression vector pCA. In an Escherichia coli strain bearing the resulting plasmid, the P protein constitutes about 20% of total soluble protein. P protein was purified using ammonium sulfate precipitation and two chromatography steps. Typical yields were 20–30 mg from 7.5 l bacterial culture. The protein is a monomer with a molecular mass of 7500 Da. The periplasmic signal peptide was processed identically in both the recombinant and the wild‐type proteins. CD spectra of both proteins were identical and indicated that the structure is highly ordered, containing approximately 80%α‐helix.

Purification in the presence of excess cysteine resulted in a form of the protein containing two reduced thiols, in agreement with the published sequence which has two cysteine residues. When cysteine was omitted from the purification buffers, no reduced thiol groups could be detected suggesting that the cysteine residues are oxidized. Both of these forms of the protein were found to bind approximately five Hg2+ ions/protein molecule in an apparently non‐specific manner. However, in the presence of external thiol compounds, the protein with reduced thiols bound only one Hg2+ ion/protein molecule with an apparent Kd of 3.7 ± 1.3 μM. Under these conditions, the protein with oxidized thiols did not bind Hg2+. The possible physiological role of this protein in Hg2+ detoxification is discussed.
Large spectral changes accompany the conformational transition of human pancreatic lipase induced by acylation with the inhibitor tetrahydrolipstatin
Authors: LÜTHI‐PENG Qiaoqian , WINKLER Fritz Karl
Published: March 2005

Human pancreatic lipase (HPL) loses more than 80% of its activity when incubated with tetrahydrolipstatin in a buffer containing bile salts. During the inactivation process, large changes are observed in intrinsic tryptophan fluorescence and in the near‐ultraviolet circular dichroism. The rate of chemical inactivation is highly comparable to that determined from the time dependence of the spectral changes. It is concluded that HPL undergoes a conformational transition upon inhibitor binding, resulting in a change in the microenvironment of tryptophan residues. Bile salts are needed in this system for effective inactivation of the enzyme by tetrahydrolipstatin, and a large increase in the inactivation rate takes place at about the critical micellar concentration (CMC) of bile salts. The inhibited enzyme can be reactivated by reducing the bile salt concentration to below the CMC, and the changes in tryptophan fluorescence induced by acylation with tetrahydrolipstatin are thereby reversed. This suggests that bile salts above their CMC stabilize the acyl‐enzyme complex.
Cloning and expression of an Arabidopsis nitrilase which can convert indole‐3‐acetonitrile to the plant hormone, indole‐3‐acetic acid
Authors: BARTLING Dieter , SEEDORF Matthias , MITHÖFER Axel , WEILER Elmar W.
Published: March 2005

From an Arabidopsis thaliana cDNA expression library, a cDNA clone was isolated, characterized and sequenced which, at the amino acid level, resembled the Klebsiella ozaenae bromoxynil nitrilase encoded by the bxn gene. The cDNA contained a long open reading frame, starting from two possible neighbouring ATG codons and capable of encoding 340 or 346 amino acids with calculated molecular masses of 37526 Da or 38176 Da, respectively. The sequence similarity between the deduced polypeptides from the Arabidopsis cDNA and bxn was clustered in three domains, one at the C‐terminus, one in the center and one near the N‐terminus of the two proteins, suggesting important functional elements in these parts of the proteins. The cDNA was cloned into different vectors under the control of the lacZ promotor and was functionally expressed by induction with isopropyl‐β‐d‐thiogalactoside. Using a combination of high‐performance liquid chromatography, monoclonal‐antibody based enzyme‐linked immunosorbent assay and mass spectroscopy, it was shown that the isolated cDNA clone encodes an enzymatically active nitrilase which is able to convert indole‐3‐acetonitrile to the plant growth hormone, indole‐3‐acetic‐acid.
Cloning of the chicken α3(IX) collagen chain completes the primary structure of type IX collagen
Authors: BREWTON Randolph G. , OUSPENSKAIA Maia V. , van der REST Michel , MAYNE Richard
Published: March 2005

Type IX collagen is composed of three genetically distinct polypeptides that contain several collagenous and non‐collagenous domains. The α2(IX) chain also contains a covalently bound glycosaminoglycan side chain. Type IX collagen is located on the surface of collagen fibrils of both hyaline cartilage and vitreous humor, such that one of the collagenous domains (COL3) projects from the surface of the fibril in a periodic manner. We have cloned and sequenced a full‐length cDNA for the chicken α3(IX) collagen chain from a cartilage cDNA library. Together with the sequence of the α1(IX) and α2(IX) chains, this completes the primary structure of type IX collagen for one species. These sequences will be useful to better understand the mechanism of triple‐helix formation in type IX collagen and the nature of type II and type IX collagen interactions in fibril formation.
A synthetic peptide encompassing the binding site of the second zinc atom (the ‘structural’ zinc) of alcohol dehydrogenase
Authors: BERGMAN Tomas , JÖRNVALL Hans , HOLMQUIST Barton , VALLEE Bert L.
Published: March 2005

A 23‐residue peptide was synthesized that incorporates the loop which binds the structural zinc atom of mammalian alcohol dehydrogenases and contributes, in part, to subunit interactions in the native enzyme. Neither the amino acid composition nor the sequence of the peptide resemble those of zinc fingers. The reduced peptide stoichiometrically binds zinc or cobalt to form stable complexes with a dissociation constant for the peptide/Co2+ complex of 2.1 μM at pH 7.5. EDTA disrupts the complex. The absorption and magnetic circular dichroic spectra of the cobalt‐peptide are indicative of a tetrahedral coordination geometry, and are similar to those of the cobalt‐substituted structural site of horse and human (β1β1) liver alcohol dehydrogenases. Consequently, the synthetic peptide can serve as a model for the metal‐binding segment of alcohol dehydrogenase and for studies of fundamental problems concerning protein/metal interactions.
Two distinct genes encode two major isoelectric forms of insecticyanin in the tobacco hornworm, Manduca sexta
Authors: LI Wancheng , RIDDIFORD Lynn M.
Published: March 2005

Insecticyanin is a blue pigment found in the epidermis and hemolymph of the tobacco hornworm, Manduca sexta. Two distinct full‐length cDNA species were isolated and shown to encode the two major isoelectric forms of insecticyanin, the a form (INS‐a) and b form (INS‐b). Sequence analysis of the two cDNA clones, pE1‐I1 and pE3‐I2, reveals that both contain an 618‐bp open reading frame which predicts an 189‐amino‐acid protein and an 17‐amino‐acid signal peptide.

Comparison of the deduced INS‐a and INS‐b proteins show 13 amino acid differences, of which six are conserved. Three amino acid substitutions are also found between the deduced INS‐b sequence and the sequenced INS‐b protein isolated from the hemolymph. Isolation and characterization of five genomic clones revealed that pE1‐I1 and pE3‐I2 come from two different genes. Both INS‐a and INS‐b genes have four exons interrupted by three introns at the same positions. The two genes share 93% nucleotide similarity in the coding region. Moreover, the ‘TATA box’ and ‘CAAT box’ are completely conserved in the putative promoter regions of the two genes. Primer extension revealed that both INS‐a and INS‐b genes begin their transcription at position – 52 relative to their translation initiation codon, ATG. We conclude that the two genes are the result of gene duplication.
Activity of heart and muscle lactate dehydrogenases in all‐aqueous systems and in organic solvents with low amounts of water
Authors: FERNÁNDEZ‐VELASCO D. Alejandro , GARZA‐RAMOS Georgina , RAMÍREZ Leticia , SHOSHANI Liora , DARSZON Alberto , DE GÓMEZ‐PUYOU Marietta TUENA , GÓMEZ‐PUYOU Armando
Published: March 2005

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of lactate dehydrogenases from heart and muscle was studied in standard water mixtures and in reverse micelles formed with n‐octane, hexanol, cetyltrimethylammonium bromide and water in a concentration that ranged over 2.5–6.0% (by vol.). In all water mixtures GdmCl (0.15–0.75 M) and urea (0.5–3.0 M) inhibited the activity of the enzymes at non‐saturating pyruvate concentrations. At concentrations of pyruvate that proved inhibitory for enzyme activity due to the formation of a ternary enzyme‐NAD‐pyruvate complex, GdmCl and urea increased the activity of the enzymes. This increase correlated with a decrease of the ternary complex, as evidenced by its absorbance at 320–325 nm. In the low‐water system it was found that: (a) at all concentrations of pyruvate tested (0.74–30 mM), GdmCl enhanced the activity of the heart enzyme to a similar extent; (b) in the muscle enzyme, GdmCl inhibited or increased the activity through a process that depended on the concentration of pyruvate and GdmCl; (c) under optimal conditions, the activation by GdmCl was about two times lower in the muscle than in the heart enzyme, although in all‐water media the activity of the muscle enzyme was twice as high. The expression of lactate dehydrogenase activity in the low‐water system was higher with the heart than with the muscle enzyme compared to their activities in all‐water media (about 260 and 600 μmol min−1 mg−1 in the heart and muscle enzymes respectively). Apparently for catalysis, the water requirement in the heart enzyme is lower than in the muscle enzyme. It is likely that the different response of the two enzymes to solvent is due to their distinct structural features.
Heat‐shock response in cultured chick embryo chondrocytes
Authors: NERI Monica , DESCALZI‐CANCEDDA Fiorella , CANCEDDA Ranieri
Published: March 2005

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows ‘in vitro’ differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi‐Cancedda, F. and Cancedda, R. (1986) J. Cell. Biol. 102, 2310–2317]. As control, we used cultures of embryonic fibroblasts from the whole body and from the skin. In the cell lysates of all cultures we identified four major heat‐shock proteins (HSP), with a molecular size corresponding to HSP families previously described (HSP 90, HSP 70, HSP 47 and HSP 26). Some of these proteins were constantly induced when the temperature was raised, others were expressed in a more variable manner. Differences also existed in the relative amount of the HSP synthesized by the four cultures.

When we specifically investigated HSP species released into the culture medium, we observed a 43–45 kDa protein constantly expressed and secreted in large amount by the cells. On the basis of its biochemical characteristic and its precipitation by specific antibodies, this protein has been identified as osteonectin (SPARC, BM‐40).
Expression of Erythrina corallodendron lectin in Escherichia coli
Authors: ARANGO Rafael , ADAR Rivka , ROZENBLATT Shmuel , SHARON Nathan
Published: March 2005

The cDNA of the Erythrina corallodendron lectin (ECorL) has been expressed in Escherichia coli. For this purpose, an NcoI site was inserted into the cDNA coding for the lectin precursor [Arango, R., Rozenblatt, S. & Sharon, N. (1990) FEBS Lett. 264, 109–112] immediately before the codon GTG (103–105) which codes for the N‐terminal valine of the mature lectin. This introduced an ATG codon for a methionine preceding the valine. The mutated cDNA was ligated into pUC‐8, then subcloned into the expression vector pET‐3d, which carries a strong promoter derived from gene 10 of the phage T7. The recombinant plasmid was introduced into the E. coli lysogenic strain BL21(DE3). Recombinant ECorL was expressed by growing the bacteria in the presence of isopropyl β‐d‐thiogalactopyranoside. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 6 M urea in cyclohexylaminopropane sulfonate pH 10.5, renaturation by 10‐fold dilution in the same buffer and further adjustment of the pH to 8.0. The recombinant lectin, obtained at a yield of 4–7 mg/1 culture, had, by gel filtration, a slightly lower molecular mass (56 kDa) than the native lectin, and was devoid of covalently linked carbohydrate; it was, however, essentially indistinguishable from native ECorL by other criteria, including its dimeric structure, Western blot analysis with anti‐ECorL polyclonal and monoclonal antibodies, and Ouchterlony double‐diffusion analysis with polyclonal antibodies, as well as hemagglutinating activity and specificity for mono‐ or disaccharides.
The complete primary structure of the boar spermadhesin AQN‐1, a carbohydrate‐binding protein involved in fertilization
Authors: SANZ Libia , CALVETE Juan José , MANN Karlheinz , SCHÄFER Wolfram , SCHMID Erich R. , TÖPFER‐PETERSEN Edda
Published: March 2005

Gamete recognition and adhesion are essential steps in the complex process of fertilization. In mammals and in other species, increasing evidence indicates that carbohydrate‐binding proteins on the sperm surface play a pivotal role as counter‐receptors for certain oligosaccharide moieties attached to the oocyte zona pellucida glycoproteins. Although different sperm‐associated zona‐pellucida‐binding proteins have been identified in a number of species, few of them have been isolated and structurally characterized. In this paper we report the primary structural characterization of AQN‐1, a 12‐kDa boar‐sperm‐associated carbohydrate‐binding and zona‐pellucida‐binding protein. The molecular mass of AQN‐1 was determined by time‐of‐flight plasma‐desorption mass spectrometry. Determination of its amino acid sequence and location of disulphide bridges were accomplished by a combination of proteochemical and mass spectrometric methods. The primary structure of AQN‐1 failed to show any significant similarity to the protein structures deposited with the Martinsried Institute for Protein Sequences data bank, indicating that it may belong to a novel protein family involved in fertilization. AQN‐1 shares extensive structural, as well as functional, similarity with two other boar sperm zona‐pellucida‐binding proteins, AQN‐3 and AWN, which we have recently characterized. To name this protein family, we have coined the term spermadhesin. Our data may be relevant for identification of spermadhesins in other species, and thus may contribute to a better understanding of the species‐specific sperm‐egg recognition mechanism.
A Streptomyces glaucescens endodeoxyribonuclease which shows a strong preference for CC dinucleotide
Authors: APARICIO Jesús F. , FREIJE José M. P. , LOPEZ‐OTIN Carlos , CAL Santiago , SANCHEZ Jesús
Published: March 2005

We have characterized a deoxyribonuclease from Streptomyces glaucescens that cleaves double‐stranded DNA preferably between the dinucleotide 5′‐CC‐3′. The cleavage specificity was demonstrated by both analysis of the terminal nucleotides of the generated fragments and DNA sequencing of partially digested DNA. Digestion of λ DNA with this enzyme resulted in the production of double‐stranded fragments with 5′ and/or 3′‐protruding single‐stranded tails. DNase I footprinting experiments indicated that the nuclease specifically binds to its cleavage sites on the DNA under noncatalytic conditions. The enzyme is not affected by cytosine methylation in hemimethylated DNA.
The iron‐sulfur‐cluster‐containing l‐serine dehydratase from Peptostreptococcus asaccharolyticus
Authors: HOFMEISTER Antje E. M. , BERGER Stefan , BUCKEL Wolfgang
Published: March 2005

The stereochemistry of the deamination of l‐threonine to 2‐oxobutyrate, catalyzed by purified l‐serine dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled l‐threonine in 2H2O and in 3HOH, as well as with l‐[3‐3H]threonine in unlabelled water. Isotopically labelled 2‐oxobutyrate thus formed was directly reduced in a coupled reaction with l‐ or d‐lactate dehydrogenase and NADH. The (2R)‐ or (2S)‐2‐hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H‐NMR spectroscopy and, after degradation of 2‐hydroxy‐butyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of l‐threonine catalyzed by l‐serine dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by serine dehydratase, therefore confirming the distinction of the l‐serine dehydratase of P. asaccharolyticus as an iron‐sulfur protein from those dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.
Chemical modification of prostaglandin endoperoxide synthase by N‐acetylimidazol
Authors: SCHERER Hans‐Josef , KARTHEIN Rainer , STRIEDER Stephan , RUF Hans Heinrich
Published: March 2005

Prostaglandin H synthase apoprotein, without its prosthetic heme group, was inactivated by N‐acetylimidazole under conditions typical for the O‐acetylation of tyrosyl residues. A spontaneous reactivation occurred above pH 7.5 at 22°C, which indicated spontaneous hydrolysis of acetylated residues. Below pH 7.5, where stable inactivation was observed, reactivation was achieved by reaction with hydroxylamine. Both enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, were inactivated and reactivated simultaneously and to the same extent. In contrast to the apoprotein, the holoenzyme with heme was not inactivated by N‐acetylimidazole. The number of acetyl groups, as determined as hydroxamate after the reaction with hydroxylamine at pH 8.2, was 2.5 ± 0.4 for the apoprotein and 1.0 ± 0.24 for the holoenzyme. The specific binding of heme as the prosthetic group was no longer observed by EPR (signals at g= 6.7 and 5.3) when hemin was added to the N‐acetylimidazole‐reacted apoprotein. Treatment of N‐acetylimidazole‐reacted apoprotein with hydroxylamine restored the specific binding of heme. The N‐acetylimidazole‐reacted apoprotein supplemented with hemin and reacted with hydroperoxides, neither showed electronic absorption spectra of higher oxidation states nor an EPR doublet signal due to a tyrosyl radical.

These results demonstrate that heme protects against the inactivating modification by N‐acetylimidazole and that this modification prevents binding of the prosthetic heme group necessary for both enzymic activities. The absence of the prosthetic heme group explains the concomitant loss of cyclooxygenase and peroxidase activities, as well as the absence of higher oxidation states and the tyrosyl radical. We suggest that the acetylation of a residue in the heme pocket, most probably a tyrosine, although a histidine cannot be definitely disproved, exerts the inhibiting effect. This residue could be the axial ligand of the heme or in close contact to the heme. The results also show that the inhibition by N‐acetylimidazole does not involve the acetylation of Ser530 which causes the inhibition by acetylsalicylic acid of cyclooxygenase. [The numbering of amino acids in ovine prostaglandin H synthase is according to DeWitt, D. L. and Smith, W. L. (1988) Proc. Natl Acad. Sci. USA 85, 1412–1416 including a signal peptide of 24 residues which is missing in the processed protein.]
Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2‐methyleneglutarate mutase from Clostridium barkeri
Authors: MICHEL Christoph , ALBRACHT Simon P. J. , BUCKEL Wolfgang
Published: March 2005

The ultraviolet/visible spectrum of the pure pink‐orange 2‐methyleneglutarate mutase from Clostridium barkeri between 300–600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen‐stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively. Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 ± 0.3 mol/mol tetrameric enzyme (m= 300 kDa). Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40–50% of the initially bound cobalamin. This preparation could be reactivated to 95–100% by addition of adenosylcobalamin. The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity. 2‐Methyleneglutarate mutase was inactivated by incubation with aquo‐, cyano‐ or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin. Upon incubation of the mutase with [5′‐3H]adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium‐labelled cofactor without loss of activity. During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin. EPR spectroscopy demonstrated the presence of 0.8 mol base‐on cob(II)alamin/mol enzyme. Upon addition of 2‐methyleneglutarate a second EPR signal of about equal intensity at g= 2.13 arose. The question of whether the oxygen‐stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation.
Inhibition of the uptake of oxidized low‐density lipoprotein in macrophage J774 by the antibiotic ikarugamycin
Authors: HASUMI Keiji , SHINOHARA Chikara , NAGANUMA Shin , ENDO Akira
Published: March 2005

The antiprotozoal antibiotic ikarugamycin was found to significantly inhibit oxidized low‐density lipoprotein(LDL)‐induced accumulation of cholesteryl ester in macrophage J774 at a concentration over 1–4 μM. Cholesteryl ester synthesis from [14C]oleate in the macrophages was similarly inhibited by the antibiotic, while the synthesis of triacylglycerol and polar lipids was not affected. The internalization of oxidized [125I]LDL in macrophages was reduced to 50% by 2 μM ikarugamycin, while cell‐surface binding of oxidized [125I]LDL, lysosomal hydrolysis of the internalized oxidized [125I]LDL and microsomal acyl‐coenzyme A: cholesterol acyltransferase was not detectably inhibited by 5 μM ikarugamycin. The results demonstrated that ikarugamycin inhibited cholesteryl ester accumulation in macrophage J774 by specifically inhibiting the uptake of oxidized LDL.
Purification and characterization of a NADH oxidase from the thermophileThermus thermophilusHB8
Authors: PARK Ho‐Jin , REISER Christian O. A. , KONDRUWEIT Simone , ERDMANN Helmut , SCHMID Rolf D. , SPRINZL Mathias
Published: March 2005

A NADH oxidase has been purified from the extreme thermophileThermus thermophilusHB8 by several chromatographic steps. The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N‐terminal amino acids sequence. It is a monomeric flavin‐adenine‐dinucleotide‐containing flavoprotein with an apparent molecular mass of 25 kDa and an 1:1 ratio of FAD to the polypeptide chain.

The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H202. The apparentKm, values for NADH and NADPH are 4.14 μM and 14.0 μM (pH 7.2 at room temperature), respectively, with a sixfold greaterkcat/Kmvalues for NADH compared to NADPH. The enzyme uses O2as an electron acceptor in the presence of either FAD, riboflavin 5′‐phosphate or riboflavin as cofactor. In addition, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (methylene blue, cytochromec, p‐nitroblue tetrazolium, 2,6‐dichloroindophenol and potassium ferricyanide), even in the absence of flavin shuttles. No significant inhibition of the NADH oxidoreductase activity by superoxide dismutase was observed with these artificial electron acceptors, indicating that electron transfer occurs mainly from NADH directly to the electron acceptors, not via O2−as an intermediate.

The purified NADH oxidase exhibits highest activity at pH 5.0 and is stable at elevated temperatures of up to 80°C.
High expression of the caprine β‐casein gene in transgenic mice
Authors: PERSUY Marie‐Annick , STINNAKRE Marie‐Georges , PRINTZ Christiane , MAHE Marie‐Françoise , MERCIER Jean‐Claude
Published: March 2005

An 18‐kb caprine genomic DNA fragment, comprising the β‐casein transcription unit with about 3‐kb 5′ and 6‐kb 3′ flanking regions, was microinjected into fertilized one‐cell murine eggs. All nine lines of transgenic mice obtained expressed the transgene in their mammary glands, as demonstrated by Northern blot analysis of mRNA in miscellaneous tissues, and qualitative and quantitative analysis of caprine β‐casein in milk, using SDS/PAGE, Western blotting and rocket immunoelectrophoresis. Two lines produced milk containing up to 21 – 24 mg of the exogenous protein/ml, a yield which is roughly twice that found in goat milk. The yield reached at least 40mg/ml in some progeny of crossbred G1 transgenic mice. Thus, the investigated gene appears to be a good candidate for making hybrid constructs that might promote an efficient production of valuable foreign proteins in milk.
Phycobilisome structure in the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC 7120
Authors: GLAUSER Manuel , BRYANT Donald A. , FRANK Gerhard , WEHRLI Ernst , RUSCONI S. Sandro , SIDLER Walter , ZUBER Herbert
Published: March 2005

Phycobilisomes of the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC7120 differ from typical tricylindrical, hemidiscoidal phycobilisomes in three respects. Firstly, size comparisons of the core‐membrane linker phycobiliproteins (LCM)in different cyanobactertia by SDS/PAGE reveal an apparent molecular mass of 120 kDa for the LCM of M. laminusus and Anabaena sp. PCC7120. This observation suggests that the polypeptides of these species have four linker‐repeat domains. Secondly, phycobilisomes of M. laminosus are shown to contain at least three, but most probably four, different rod‐core linker polypeptides (LRC). These LRC, which attach the peripheral rods to the core and thereby make phycocyanin/allophycocyanin contacts, have been identified and characterized by N‐terminal amino acid sequence analysis. Additionally, electron microscopy of phycobilisomes isolated from M. laminosus and Anabaena sp. PCC7120 reveals similar structures which differ from those of Calorthrix sp. PCC7601 with their typical six, peripheral rods. Based upon protein‐analytical results and a reinterpretation of the data of [Isono, T. & Katoh, T. (1987) Arch. Biochem. Biophys. 256, 317–324], we discuss structural implications of recent findings on the established hemidiscoidal model for the phycobilisomes of M. laminosus and Anabaena sp. PCC7120. Up to eight peripheral rods are suggested to radiate from a modified core substructure which contains two additional peripheral allophycocyanin hexamer equivalents that serve as the core‐proximal discs for two peripheral rods.
Carbohydrate structure of a thrombin‐like serine protease from Agkistrodon rhodostoma
Authors: PFEIFFER Günter , DABROWSKI Ursula , DABROWSKI Janusz , STIRM Stephan , STRUBE Karl‐Hermann , GEYER Rudolf
Published: March 2005

The carbohydrate side chains of the thrombin‐like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic glycopeptides by treatment with peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F and fractionated by high‐performance liquid chromatography. Glycans obtained were characterized by digestion with exoglycosidases, methylation analysis and, in part, by liquid secondary‐ion mass spectrometry and 1H‐NMR spectroscopy.

The results reveal that this snake venom glycoprotein contains partially truncated di‐, tri‐ and tetraantennary complex type N‐glycans carrying Fuc(α1‐6) residues at the innermost N‐ acetylglucosamine and solely (α2‐3)‐linked sialic acid substituents. As a characteristic feature, ancrod oligosaccharides comprise mainly sialylated Galβ3GlcNAcβ lactosamine antennae. Furthermore, a small proportion of the sugar chains were found to carry a NeuAcα3GalNAcβ4GlcNAcβ antenna exclusively linked to C‐2 of Man(α1‐3) residues of the pentasaccharide core. Thus, many of the glycans found represent novel glycoprotein‐N‐glycan structures.
Adenosine(5′)hexaphospho(5′)adenosine stimulation of a Ca2+‐induced Ca2+‐release channel from skeletal muscle sarcoplasmic reticulum
Authors: MORII Hiroshi , MAKINOSE Madoka
Published: March 2005

Stimulation of a Ca2+‐induced Ca2+‐release channel from skeletal muscle sarcoplasmic reticulum by various adenosine(5′)oligophospho(5′)adenosines (ApnA, n= 2–6) by a rapid quenching technique using radioactive calcium was studied. Ap4A, Ap5A and Ap6A, as well as adenosine 5′‐[β, γ‐methylene]triphosphate (AdoPP [CH2]P), a non‐hydrolyzable ATP analogue, stimulated the Ca2+‐release channel, whereas Ap2A and Ap3A had no effect. At a concentration of 0.5 mM, the order of stimulation was AdoPP[CH2]P < Ap4A < Ap5A ≪ Ap6A. As well as having the highest affinity (0.44 mM for half‐maximal stimulation), Ap6A showed an extraordinarily high Hill coefficient of 3.3 (1.9 for AdoPP[CH2]P, 2.1 for Ap5A). The stimulating effect of Ap6A was reversible, yet its dissociation proceeded very slowly. Stimulation of Ca2+ release by Ap6A was counteracted by Mg2+ and ruthenium red. A 2′,3′‐dialdehyde derivative of Ap6A, which is a chemical probe for amino groups, stimulated irreversibly the Ca2+‐release channel and modified some high‐molecular‐mass sarcoplasmic reticulum proteins, possibly including the channel protein. Our data suggest that Ap6A stimulates the Ca2+ channel by binding to the activation site of the channel subunit and simultaneously preventing the spontaneous decay of the Ca2+ channel by keeping together two of the four channel subunits by bridging them with its two adenosine groups.
Inhibition of lipoxygenase activity in lentil protoplasts by monoclonal antibodies introduced into the cells via electroporation
Authors: MACCARRONE Mauro , VELDINK Gerrit A. , VLIEGENTHART Johannes F. G.
Published: March 2005

The isolation of lentil protoplasts and the transfer of anti‐lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporated monoclonal antibody. Moreover, the inhibition of lipoxygenase activity was well correlated with the increase of protoplast viability.
Purification of a glucose‐binding protein from rat liver nuclei
Authors: SCHRÖDER Heinz C. , FACY Patrice , MONSIGNY Michel , PFEIFER Karin , BEK Andreas , MÜLLER Werner E. G.
Published: March 2005

A nuclear carbohydrate‐binding protein with a molecular mass of 67 kDa (CBP67), which is specific for glucose residues, was purified to essential homogeneity from rat liver nuclear extracts. This protein could also be isolated from nuclear ribonucleoprotein (RNP) complexes by extraction in the presence of 0.6 M or 2 M NaCl, but it was absent in polysomal RNP complex. The binding of the purified protein, which has an isoelectric point of 7.3, to glucose‐containing glycoconjugates depends on the presence of Ca2+ and Mg2+. Using closed nuclear envelope vesicles as a system to study nuclear transport of RNA, it was shown that both entrapped polysomal mRNA and nuclear RNA precursors are readily exported from the vesicles in an ATP‐dependent manner. The transport was unidirectional and strongly promoted by the poly(A) segment attached to these RNAs. In contrast, nuclear RNP complexes entrapped into the vesicles together with glucose‐conjugated bovine serum albumin or nucleoplasmin, or bird nest glycoprotein, were not exported into the extravesicular space. However, transport of nuclear RNP complexes could be achieved in the presence of glucose or after co‐addition of a glucose‐recognizing lectin from Pellina semitubulosa. In Western blots, radioiodinated CBP67 binds to an 80‐kDa polypeptide both in isolated rat liver nuclear envelopes and pore‐complex laminae. From these results we postulate that CBP67 may direct nuclear RNP complexes to the nuclear pore.
Molecular characterization of the genomic regions of the Drosophilaα‐type submit proteasome genes PROS‐Dm28.1 and PROS‐Dm35
Authors: FRENTZEL Stefan , TROXELL Megan , HAASS Christian , PESOLD‐HURT Brigitte , GLÄTZER Karl Heinz , KLOETZEL Peter‐M.
Published: March 2005

The proteasome (multicatalytic proteinase) consists of a large number of non‐identical protein subunits which are encoded by the evolutionarily conserved PROS gene family. Using the PROS‐Dm35 and PROS‐Dm28.1 cDNAs as probes, we have isolated the corresponding genomic DNA clones of Drosophila melanogaster. In situ hybridization shows that the members of the PROS gene family are not organized in a single gene cluster and that, in contrast to the PROS‐Dm35 gene, the PROS‐Dm28.1 gene is localized on the X chromosome. Analysis of the genomic organization of the PROS‐Dm28.1 and PROS‐Dm35 genes reveals that both genes are interrupted by two small introns whereby the relative positions of the introns within the two coding regions are not conserved. Neither gene possesses a distinct transcriptional start site as shown by nuclease S1 analysis. Since the promoter regions also do not contain a TATA box, PROS genes appear to be typical house‐keeping genes. A putative heat‐shock element in the promoter region of the PROS‐Dm35 gene was shown to be inactive on stress induction when fused to a reporter gene and tested in transient transfections assays. In addition, promoter deletion analysis demonstrates that the promoter region between positions –605 and –330 contains sequence elements important for PROS‐Dm35 gene activity and that deletions beyond position –150 result in an almost complete inhibition of transcription.
Characterization and crystal structure of zinc azurin, a by‐product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin
Authors: NAR Herbert , HUBER Robert , MESSERSCHMIDT Albrecht , FILIPPOU Alexander C. , BARTH Manfred , JAQUINOD Michel , van de KAMP Mart , CANTERS Gerard W.
Published: March 2005

Azurin*, a by‐product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X‐ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo‐protein after its expression and transport into the periplasm. Holo‐azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copper ions.

An X‐ray crystallographic analysis of azurin* at 0.21‐nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co‐ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co‐ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45.

The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, whch is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.
Presteady‐state and steady‐state kinetic properties of human cytochrome c oxidase
Authors: Van KUILENBURG André B. P. , GORREN Anton C. F. , DEKKER Henk L. , NIEBOER Popko , Van GELDER Bob F. , MUIJSERS Anton O.
Published: March 2005

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady‐state and steady‐state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase.

The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady‐state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur.

The proposed model was also used to explain the steady‐state kinetic parameters under various conditions. At high ionic strength (I= 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate‐limiting step.

The kcat value of the ‘high‐affinity’ phase, observed at low ionic strength (I= 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate‐limiting step in the ‘high‐affinity’ phase at low ionic strength.

The ‘low‐affinity’kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron‐transfer rate of the ‘low‐affinity’ reaction is enhanced by the binding of a second molecule of cytochrome c.
Purification and membrane topology of PSI‐D and PSI‐E, two subunits of the photosystem I reaction center
Authors: LAGOUTTE Bernard , VALLONI Olivier
Published: March 2005

Structural studies have been conducted on polypeptides PSI‐D and PSI‐E, whch are extrinsic but firmly bound to the photosystem I reaction center. These subunits are predicted to be involved in the correct interaction with soluble electron acceptor(s), like ferredoxin. We designed an original method to extract both polypeptides directly from thylakoid membranes and to purify them: a stepwise extraction with NaSCN followed by size fractionation and reverse‐phase HPLC. Investigation of the in situ topology of PSI‐D and PSI‐E was undertaken using monoclonal antibody binding, controlled proteolysis, peptide sequencing and electron microscopy. The precise identification of numerous proteolytic sites indicates that the entire N‐terminal regions of PSI‐E (up to Glu15) and PSI‐D (up to LyslS) are exposed to the medium. Partial mapping of the exposed epitopes was possible using purified fragments of each polypeptide. In the case of PSI‐E, this mapping confirmed the accessibility of the N‐terminal part, and suggested the need for another exposed sequence, probably located after Met39 in the second half of the protein. For PSI‐D, this mapping revealed that the sequence between Met74 and Metl40, including the most basic amino acid clusters, is also partly accessible. These experiments provide the first detailed informations, although still partial, on the topology of these polypeptides. They give a preliminary basis for hypotheses concerning the sites of interaction with the soluble counterparts.
Increased steady‐state levels of several mitochondrial and nuclear gene transcripts in rat hepatoma with a low content of mitochondria
Authors: LUCIAKOVÁ Katarína , KUŽELA Štefan
Published: March 2005

Cells from a rapidly growing rat Zajdela hepatoma were shown to contain (on a protein basis) five‐times less mitochondria than hepatocytes from resting or regenerating rat liver. Transcripts of four nuclear genes for representative mitochondrial membrane proteins (β‐F1 subunit and N,N′‐dicyclohexyl‐carbodimide‐binding protein of ATP synthase, subunit IV of cytochrome oxidase and ADP/ATP translocase) were present in 2–4‐times higher amounts in the poly(A)‐rich RNA of the hepatoma than in the corresponding RNA fraction from resting or regenerating rat liver. The liver and hepatoma transcripts for the β‐F1, subunit were translated in an in‐vitro system with equal efficiency. Pulse‐chase labeling of isolated Zajdela hepatoma cells and hepatocytes from resting and regenerating liver revealed a relative excess of the newly synthesized β‐F1 subunit in the tumor cells. The half‐life of the β‐F1, subunit was significantly shorter in the hepatoma cells than in hepatocytes from resting and regenerating liver. The contents of transcripts of three mitochondrial genes examined (cytochrome oxidase subunits I and II and NADH‐ubiquinone reductase subunit 2) in Zajdela hepatoma mitochondria were about five‐times higher than in the mitochondria of the resting cells and 3–4 times higher than in the organelles of the regenerating organ. The results indicate that events other than transcription (most likely post‐translational) may be responsible for the reduced content of mitochondria in tumor cells.
Specific inhibition of Physarum polycephalum DNA‐polymerase‐α‐primase by poly(l‐malate) and related polyanions
Authors: HOLLER Eggehard , ACHHAMMER Gunthar , ANGERER Bernhard , GANTZ Birgit , HAMBACH Christoph , REISNER Hermine , SEIDEL Bettina , WEBER Cornelia , WINDISCH Christina , BRAUD Christian , GUERIN Philipe , VERT Michel
Published: March 2005

Poly(l‐malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(l‐malate) and structurally related polyanions can interact with DNA‐polymerase‐α‐primase complex and with histones of P. polycephalum. Poly(l‐malate) is found to inhibit the activities of the DNA‐polymerase‐α‐primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(l‐malate), and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(l‐malate), poly(d,l‐malate), poly(l‐aspartate), poly(l‐glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(l‐malate) (N= 5) and poly(d,l‐malate) (N= 5) are the most efficient inhibitors, followed by poly(l‐aspartate) (N= 6), poly(acrylate) (N= 3), poly(l‐glutamate) (N= 8), poly(vinyl sulfate) (N= 3). It is proposed that poly(l‐malate) interacts with DNA‐polymerase‐α‐primase of P. polycephalum. According to its physical and biochemical properties, poly(l‐malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock‐piling agent of DNA‐polymerase‐α‐primase in the G2 phase and M phase of the plasmodial cell cycle.
Eukaryotic DNA primase appears to act as oligomer in DNA‐polymerase‐α–primase complex
Authors: PODUST Vladimir N. , VLADIMIROVA Olga V. , MANAKOVA Elena N. , LAVRIK Olga I.
Published: March 2005

Human placenta and calf thymus DNA‐polymerase‐α–primases were analyzed using native gradient‐polyacrylamide‐gel electrophoresis followed by overlay assays of polymerase and primase activities. The human enzyme contained three catalytically active native forms of 330, 440 and 560 kDa and the bovine enzyme five forms of 330, 440, 500, 590 and kDa. Of the various DNA polymerase forms. only the largest (560 kDa for human DNA polymerase and 590 kDa and 660 kDa for bovine DNA polymerase) contained primase activity. Titration of human DNA‐polymerase‐α–primase with DNA‐polymerase‐free primase caused the conversion of the 440‐kDa to the 560‐kDa form. The data favour the idea that primase binds to DNA polymerase α as an oligomer of 3 primases/polymerase core.

In addition, the ability of primase to utilize oligoriboadenylates containing (prA)n or pp(prA)n was investigated. The primase elongated pp(prA)2 – 7 up to nanoadenylates or decaadenylates, but did not add 9 or 10 mononucleotides to a preexistent primer. In contrast to pp(prA)n < 10, (prA)n < 10 were rather poor primers for the primase. Both pp(prA)8,9 and (prA)n > 10 were elongated by primase, producing characteristic multimeric oligonucleotides. The possible connection of the structure of the DNA‐polymerase‐α–primase complex with the catalytical properties of primase is discussed.
Multiphasic denaturation of the λ repressor by urea and its implications for the repressor structure
Authors: BANIK Utpal , SAHA Rina , MANDAL Nitai C. , BHATTACHARYYA Bhabatarak , ROY Siddhartha
Published: March 2005

Urea denaturation of the λ repressor has been studied by fluorescence and circular dichroic spectroscopies. Three phases of denaturation could be detected which we have assigned to part of the C‐terminal domain, N‐terminal domain and subunit dissociation coupled with further denaturation of the rest of the C‐terminal domain at increasing urea concentrations. Acrylamide quenching suggests that at least one of the three tryptophan residues of the λ repressor is in a different environment and its emission maximum is considerably blue‐shifted. The transition in low urea concentration (midpoint approximately 2 M) affects the environment of this tryptophan residue, which is located in the C‐terminal domain. Removal of the hinge and the N‐terminal domain shifts this transition towards even lower urea concentrations, indicating the presence of interaction between hinge on N‐terminal and C‐terminal domains in the intact repressor.
Neuropeptide Y
Authors: MIERKE Dale F. , DÜRR Hansjörg , KESSLER Horst , JUNG Günther
Published: March 2005

The 36‐amino‐acid neuropeptide Y (human), which is one of the most potent vasoconstrictors and which exhibits a number of other biological functions, has been synthesized using automated peptide synthesis. The optimized method, using 9‐fluorenylmethoxycarbonyl protecting and single‐step coupling, yielded the crude product in 90% purity allowing for single‐step reversed‐phase HPLC purification to >98% purity and a high overall yield (50%). The hormone was characterized by several chromatographic methods, ion‐spray mass spectroscopy and Edman degradation. The conformation of human neuropeptide Y was examined by CD, NMR and computer simulations. The CD measurements in trifluoroethanol/water (9:1) show a large percentage of α‐helix. Variation of concentration, from 0.5 μM increasing up to the 1 mM used for NMR measurements, indicates no evidence for aggregation. In the same solvent system, the NMR line widths were very broad and therefore the resonance assignment was achieved with the exclusive use of two‐dimensional NOE spectra. The 248 clearly distinguishable NOEs from the NMR study were used in distance geometry calculations and the resulting structures were refined with restrained molecular dynamics. The results indicate an α‐helix extending from Arg19 to Gln34. For the N‐terminal half of the molecule no regular structure was observed.