bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of CYP2D6 in developing human liver
Authors: TRELUYER Jean‐Marc , JACQZ‐AIGRAIN Evelyne , ALVAREZ Fernando , CRESTEIL Thierry
Published: March 2005

The CYP2D6 protein is a polymorphic isoenzyme involved in the biotransformation of several drugs including the probe drug dextromethorphan. The rise in the protein concentration, immunochemically determined with a specific antibody, was shown to occur within the first week following birth, whatever the gestational age at birth.

In fetuses, the concentration of hepatic CYP2D6 protein was very low or undetectable in 70% of samples tested. In the remaining 30%, its concentration was comparable to that of newborns aged 1–7 days. This early rise was associated with spontaneous abortion in 70% of positive samples, whereas in fetuses with an intermediate CYP2D6 protein concentration, 80% were from induced abortions.

The rise in CYP2D6 protein was associated with the developmental onset of dextromethorphan O‐demethylation, but not N‐demethylation, even if activity was lower in fetal than in neonatal and in adult liver microsomes.

Lastly, the CYP2D6 RNA is detectable earlier than the protein and exhibits a peak of hepatic accumulation in newborns, before declining in adulthood. A positive correlation between RNA accumulation and protein concentration can be demonstrated only in the adult. This suggests that regulation is primarily at the transcriptional level, but cannot rule out the participation of post‐transcriptional events in the regulation process throughout ontogenesis.
Mollusc‐specific toxins from the venom of Conus textile neovicarius
Authors: FAINZILBER Michael , GORDON Dalia , HASSON Arik , SPIRA Micha E. , ZLOTKIN Eliahu
Published: March 2005

Three peptide toxins exhibiting strong paralytic activity to molluscs, but with no paralytic effects on arthropods or vertebrates, were purified from the venom of the molluscivorous snail Conus textile neovicarius from the Red Sea. The amino acid sequences of these mollusc specific toxins are: TxIA, WCKQSGEMCNLLDQNCCDGYCIVLVCT (identical to the so called ‘King Kong peptide’); TxIB, WCKQSGEMCNVLDQNCCDGYCIVFVCT; TxIIA, WGGYSTYCγVDSγCCSDNCVRSYCT (γ=γ‐carboxyglutamate).

There is a similarity of the Cys framework of these toxins to that of the ω‐conotoxins; however, their net negative charges, high content of hydrophobic residues and uneven number of Cys residues in TxIIA, are highly unusual for conotoxins. When assayed on isolated cultured Aplysia neurons, all three toxins induced membrane depolarization and spontaneous repetitive firing. The TxI toxins also induce a marked prolongation of the action potential duration, which is sodium dependent. These effects differ significantly from the blocking activities of piscivorous venom conotoxins. These mollusc specific conotoxins may therefore serve as new and selective probes for ion‐channel functions in molluscan neuronal systems.
Site‐directed mutagenesis and epitope‐mapped monoclonal antibodies define a catalytically important conformational difference between human placental and germ cell alkaline phosphatase
Authors: HOYLAERTS Marc F. , MILLÁN José Luis
Published: March 2005

Placental (PLAP) and germ cell (GCAP) alkaline phosphatases were probed immunologically with a library of 18 murine monoclonal antibodies reacting with different conformational epitopes on PLAP. Three main antigenic domains (I, II and III) were mapped by antibody competition experiments and the relative binding of the antibodies to site‐directed PLAP mutants. Relative affinities of each of the antibodies for the wild type (wt) GCAP were 2–3‐fold lower than the values found for wt PLAP. Relative affinity was determined for a series of PLAP mutants, in which one, two or three amino acids were substituted for the corresponding wt GCAP residues by site‐directed mutagenesis. Substitutions at residues 15, 38, 67, 241 or 254 induced a major decrease in affinity (6–10‐fold) primarily for those antibodies reacting within domain I, whereas changes at positions 84 and 297 led to a 2–3‐fold enhancement of affinities as measured with antibodies reacting within the three domains. Arg209 was found to constitute the only difference between the S and F allelic phenotypes of PLAP and to structure the epitope for the F/S allotype‐discriminating antibodies. Arg241 was found to constitute the epitope for the antibody 17E3 that discriminates between PLAP and GCAP. Mutagenesis at position 68 or 133 had little effect on the overall reactivity with the antibody panel. Substitution in wt PLAP of Glu429 for Gly429 or even for His429 (found at this position in tissue‐nonspecific alkaline phosphatase) and Ser429 (found in the intestinal alkaline phosphatase) induced a general decrease in affinities as detected by 16 of the 18 antibodies. The conformational change accompanying mutagenesis of Glu429 in PLAP, is important in view of the recent identification of Gly429 as the major determinant of the unique GCAP inhibition by the uncompetitive inhibitor l‐Leu Relative affinity values determined for the rare l‐Leu sensitive heterodimeric FD and SD PLAP phenotypes, suggested that the reactivity pattern of the D homodimer with the antibody panel, would resemble more closely that of wt GCAP than wt PLAP. Our data suggest that the uncompetitive inhibition of GCAP by l‐Leu is due to an enzymatically critical conformational change in a loop region proximal to the active site of the enzyme, induced by substitution of a single amino acid residue.
Complete primary structure of porcine tenascin
Authors: NISHI Tatsunari , WEINSTEIN Jasminder , GILLESPIE William M. , PAULSON James C.
Published: March 2005

Tenascin is an extracellular matrix protein that is postulated to modulate tissue differentiation and cell migration during development. cDNA clones for tenascin were isolated from a cDNA library of adult porcine submaxillary glands. Three forms of tenascin clones were observed which varied with the number (8–10) of fibronectin type III (FN‐III) domains. A major form consists of the N‐terminal domain involved in the hexamer formation of tenascin subunits, 14 epidermal‐growth‐factor‐like domains, nine FN‐III domains, and the fibrinogen‐like domain. A minor form with ten FN‐III domains has never been described. Another striking feature is the lack of an RGD sequence that has been implicated to be crucial for cell adhesion, whereas RGD is present in both chicken and human tenascin sequences. In the adult, tenascin is expressed in very restricted tissues such as brain and chicken gizzard. A survey of tenascin transcripts in various adult rat normal tissues, including brain, revealed that the transcripts were detected only in submaxillary glands where tenascin expression has never been reported.
Activation of O2−‐generating oxidase in an heterologous cell‐free system derived from Epstein‐Barr‐virus‐transformed human B lymphocytes and bovine neutrophils
Authors: COHEN‐TANUGI Laurence , MOREL Françoise , PILLOUD‐DAGHER Marie‐Claire , SEIGNEURIN Jean Marie , FRANCOIS Patrice , BOST Michel , VIGNAIS Pierre V.
Published: March 2005

Epstein‐Barr‐virus‐transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4β‐phorbol 12‐myristate 13‐acetate exhibit an NADPH‐dependent oxidase activity capable of generating the superoxide anion O2−, similar to, but less efficient than that of activated neutrophils. A cell‐free system of oxidase activation consisting of a membrane fraction and cytosol from EBV B lymphocyte homogenate supplemented with guanosine 5′‐[γ‐thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O2−, assessed by the superoxide‐dismutase‐sensitive reduction of cytochrome c in the presence of NADPH. However, cytochrome c reduction was slow and largely insensitive both to superoxide dismutase, and to iodonium biphenyl, a powerful inhibitor of the oxidase activity in neutrophils. A markedly faster eduction of cytochrome c in the presence of NADPh was obtained with a heterologous system consisting of cytosol from EBV B lymphocytes and bovine neutrophil membranes, GTP[S], arachidonic acid and Mg2+; in this system, reduction of cytochrome c was totally inhibited by superoxide dismutase and iodonium biphenyl. These results show that EBV B lymphocytes contain a substantial amount of cytosolic factors of oxidase activation, and that the limiting factors for O2− production in B lymphocytes are the membrane components of the oxidase complex. The heterologous system of EBV B lymphocyte cytosol and bovine neutrophil membranes provided a rapid and convenient method to diagnose cytosolic defects in autosomal forms of chronic granulomatous disease. In addition, it might be a useful tool to explore the mechanism of action of the cytosolic factors in oxidase activation.
Purification and characterization of the cytosolic NADP+‐dependent malic enzyme from human breast cancer cell line
Authors: CHANG Gu‐Gang , WANG Jehng‐Kang , HUANG Ter‐Mei , LEE Hwei‐Jen , CHOU Wei‐Yuan , MENG Ching‐Liang
Published: March 2005

Cytosolic NADP+‐dependent malic enzyme from a cultured human breast cancer cell line was purified to near homogeneity by two highly efficient chromatography systems: Pharmacia‐LKB Q‐Sepharose anion‐exchange chromatography and adenosine‐2′,5′‐bisphosphate–agarose affinity chromatography. Teh overall yield was 27%. The enzyme is presumably a tetramer composed of four probably identical subunits of Mr 65000, which is similar to the enzyme from other sources. The pI and optimum reaction pH values for the tumor malic enzyme are 5.5 and 7.2, respectively. At pH 6.9, most of the enzyme exists as monomers. Activation energy for the enzyme‐catalyzed oxidative‐decarboxylation reaction is 57.4 kJ/mol. The enzyme is strictly NADP+ dependent, as NAD+ cannot support the oxidative‐decarboxylation reaction. ATP at low concentration inhibits the enzyme activity. Fumarate at concentrations up to 5 mM does not affect the enzymatic reaction rate. Therefore the tumor cytosolic malic enzyme, unlike the mitochondrial malic enzyme, is not an allosteric regulatory enzyme.
αB‐crystallin exists as an independent protein in the heart and in the lens
Authors: BHAT Suraj P. , HORWITZ Joseph , SRINIVASAN Anupama , DING Linlin
Published: March 2005

αB‐crystallin, a polypeptide of molecular mass 22 kDa, is considered to be one of two subunits (αA and αB) of the multimeric lens‐specific protein, α‐crystallin. Recent demonstrations of the extralenticular presence of αB‐crystallin have suggested that outside of the lens, this polypeptide may have functions independent of αA. Within the lens however, as part of the protein α‐crystallin, its function is assumed to be structural. In an effort to investigate the functional status of αB‐crystallin in the lens, we have characterized this polypeptide in the rat heart and the human lens.

Unequivocal identity of αB‐crystallin in the rat heart and the rat lens was established by the sequence analyses of the respective cDNA clones. Size exclusion chromatography (FPLC) and immunoblotting showed that in the rat heart, αB‐crystallin exists as an aggregate of 300–400 kDa average molecular mass, similar to that of purified αB‐crystallin isolated from bovine lens. Interestingly, analysis of the human lens proteins by immunoblotting showed that, with age, unlike αA‐crystallin, the αB subunit remains detectable in the soluble fractions derived from normal lenses as old as 82 years. Importantly, the average molecular mass of the αB subunit in the soluble fractions prepared from 60–80‐year‐old human lens nuclei was also found to be 300–400 kDa. These data lead to the conclusion that αB‐crystallin may exist as an independent protein not only in non‐lens tissues (e.g. heart) but in the lens as well.
Phorbol esters inhibit the activity of the chicken acetylcholine receptor α‐subunit gene promoter
Authors: LAUFER Ralph , KLARSFELD André , CHANGEUX Jean‐Pierre
Published: March 2005

Protein kinase C has previously been implicated in the regulation of chicken acetylcholine receptor (AChR) gene expression. To investigate the molecular basis of this regulation, the promoter of the AChR α‐subunit (αAChR) gene was linked to a reporter gene and introduced into cultured chick myotubes by transient transfection. Treatment of myotubes with protein‐kinase‐C‐activating phorbol esters was found to inhibit promoter activity. These inhibitory actions were mediated by promoter sequences between nucleotides – 110 and – 45, relative to the start point of transcription of the αAChR gene. In particular, phorbol‐ester responsiveness could be conferred by a short DNA sequence that contains one of the two MyoD binding sites of the αAChR gene muscle‐specific enhancer. 12‐O‐Tetradecanoylphorbol 13‐acetate was found to inhibit rapidly and potently the expression of mRNAs coding for the myogenic regulators CMD1 and myogenin. Moreover, its inhibitory effect on the αAChR gene promotor could be attenuated by cotransfection of a MyoD1 expression vector. These results provide a molecular basis for the previously demonstrated involvement of protein kinase C in the regulation of αAChR biosynthesis. In addition, they lend further support to the notion that myogenic proteins play an important role in the control of αAChR gene expression.
Characterization of SH groups in porin of bovine heart mitochondria
Authors: De PINTO Vito , Al JAMAL Jalal Ahmad , BENZ Roland , GENCHI Giuseppe , PALMIERI Ferdinando
Published: March 2005

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., Götz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989)Biol. Chem. Hoppe‐Seyler 370, 1265–1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl‐alkylating agentsN‐[14C]ethylmaleimide, eosin‐5‐maleimide andN‐(1‐pyrenyl)‐maleimide. Affinity chromatography of bovine heart porin was performed with cysteine‐specific material. The results can be summarized as follows:

(1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 35 kDa form. The oxidized forms only appear after denaturation with SDS.

(2) The 35‐kDa reduced and the 33.5‐kDa oxidized forms of porin show the same pore‐forming properties after reconstitution of the protein into lipid bilayer membranes.

(3) Labelling of cysteines by eosin‐5‐maleimide andN‐(1‐pyrenyl)‐maleimide suggested their location at a boundary between the water‐phase and the lipid‐phase. Incubation of intact mitochondria withN‐ethylmaleimide prior to eosin‐5‐maleimide andN‐(1‐pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive toN‐ethylmaleimide binding.

(4) Bovine heart mitochondrial porin covalently bound to Affi‐Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl‐Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.
Expression of the wild‐type and mutated vacuolar storage protein phaseolin in Xenopus oocytes reveals relationships between assembly and intracellular transport
Authors: CERIOTTI Aldo , PEDRAZZINI Emanuela , FABBRINI Maria Serena , ZOPPE Monica , BOLLINI Roberto , VITALE Alessandro
Published: March 2005

The role played by subunit assembly in the intracellular transport of the bean storage protein phaseolin, a soluble trimeric glycoprotein, was investigated using Xenopus oocytes injected with RNA. We show that phaseolin assembly is dependent upon the level of synthesis of the protein and is required for intracellular transport out of the endoplasmic reticulum. We also show that a fraction of the assembled phaseolin is permanently retained in a post‐endoplasmic reticulum compartment. Deletion of the C‐terminal α‐helical domain fully prevents in vivo assembly but not endoplasmic reticulum retention. This indicates that this domain is necessary for trimerization but not for interactions of unassembled subunits with endoplasmic reticulum components. The truncated phaseolin has high in vivo stability. The potential implications of these findings on the possibility to improve the nutritional value of phaseolin through genetic engineering are discussed.
Arabidopsis thaliana H1 histones
Authors: GANTT J. Stephen , LENVIK Todd R.
Published: March 2005

We have isolated two Arabidopsis thaliana cDNA clones that encode different H1 histone proteins. The H1–1 and H1–2 proteins are 274 and 273 amino acids in length, respectively, Unlike the H1 histones within a single animal species, the two plant H1 proteins share little sequence similarity outside the protein's central globular domain. Within the globular domain, a pentapeptide that is extremely well conserved in animal H1 histones, is not found in either of the plant proteins. Southern blot analysis suggests that A. thaliana has only three H1 histone genes. A genomic clone encoding the H1‐1 protein was isolated and the protein‐coding region was found to consist of two exons separated by a 104‐bp intron. The site of transcriptional initiation of the H1‐1 gene was mapped by primer‐extension analysis and a conserved octamer motif, identical to that observed in most plant core histone gnes that have been characterized to date, was found 101 nucleotides upstream of the presumed transcription‐initiation site. The 3′ portion of the gene encoding H1–2 was also isolated and sequenced. When the 3′‐flanking regions of the two H1 genes were compared, several highly conserved sequences were observed that might be convergently transcribed relative to the histone genes.
The conformational stability of the redox states of lipoamide dehydrogenase from Azotobacter vinelandii
Authors: van BERKEL Willem. J. H. , REGELINK Annette G. , BEINTEMA Jaap J. , de KOK Arie
Published: March 2005

The conformational stability of holo‐lipoamide and apo‐lipoamide dehydrogenase from Azotobacter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, tm= 80°C. The thermal stability of the holoenzyme drastically decreases upon reduction. Unlike the oxidized and lipoamide two‐electron reduced enzyme species, the NADH four‐electron reduced enzyme is highly sensitive to unfolding by urea. Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride. Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two‐state mechanism. The oxidized and two‐electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu‐C. Upon cleavage of the apoenzyme or four‐electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass > 40 kDa) are transiently produced. Sequence studies show that limited trypsinolysis of the NADH‐reduced enzyme starts mainly at the C‐terminus of Arg391. In the apoenzyme, limited proteolysis by endoproteinase Glu‐C starts from the C‐terminus at the carboxyl ends of Glu459 and/or Glu435. From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface. Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization. It is concluded that reduction of lipoamide dehydrogenase to the four‐electron reduced state(s) is accompained by conformational changes promoting subunit dissociation.
Redox and flavin‐binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough)
Authors: CURLEY G. Paul , CARR Mary C. , MAYHEW Stephen G. , VOORDOUW Gerrit
Published: March 2005

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3–4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild‐type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N‐terminal methionine residue.

Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is –143 mV (25°C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is –440 mV. The effects of pH on the observed potentials were examined; E2 varies lineraly with pH (slope =–59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox‐linked protonation of the flavodoxin hydroquinone.

D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25°C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd= 0.72 μM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25°C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1=–262 mV, E2=–193 mV; 25°C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.
Physical characterization and sequence identification of the ovary maturating parsin
Authors: GIRARDIE Josiane , RICHARD Olivier , HUET Jean‐Claude , NESPOULOUS Claude , VAN DORSSELAER Alain , PERNOLLET Jean‐Claude
Published: March 2005

A novel neurohormone, which anticipates ovarian maturation, was recently purified using liquid chromatography from the African locust nervous corpora cardiaca. Both its function and production by the pars intercerebralis of Locusta migratoria lead to its name, the ovary maturating parsin (Lom OMP). In this study, the Lom OMP was physically and chemically characterized. Its multiply charged ion spectrum was interpreted as two peaks of quite equal size having molecular masses of 6923.4 Da (major peak) and 6907.3 Da. The Lom OMP presented no periodic secondary structure according to the far ultraviolet circular dichroism spectrum obtained. It is composed of 65 amino acids and included a high concentration of alanine but is devoid of cysteine, isoleucine, methionine, lysine and threonine. The amino acid sequence indicated only one microheterogeneity, observed at position 26, consisted in the replacement of serine by alanine. The calculated Mr of the two acidic isoforms (calculated pHi= 4.87) were found to be in agreement with mass spectrometry measurements. When compared to the sequence libraries, the Lom OMP, the first insect gonadotropic neurohormone, was revealed as an unique protein.
A comparison of the reactivity and stability of wild type and His388 Gln mutant phosphoglycerate kinase from yeast
Authors: JOHNSON Christopher M. , COOPER Alan , BROWN Alistair J. P.
Published: March 2005

A variety of physico‐chemical techniques have been used to probe the possible interactions between the characteristic structural domains of yeast phosphoglycerate kinase by comparison of the wild‐type enzyme with the specific H388Q mutant in which a potential interaction between His388 and Glu190 in the crucial interdomain region is disrupted. Enzyme kinetic studies indicate that, despite being structurally remote from the active site, this mutation has significant effects on both the Vmax and Km values for various substrates. The single cysteine residue in the N domain of the protein is markedly more reactive in the mutant, and this enhanced accessibility is moderated by binding of substrates and various anions. Differences are also observed in the near‐ultraviolet CD spectra of these proteins. The chemical and thermal stability of the mutant enzyme is reduced, as indicated from guanidinium chloride and differential‐scanning calorimetry denaturation studies. Moreover, interdomain interactions seem to be altered in the mutant, resulting in the appearance of independent thermal transition for the two domains, in contrast to the single cooperative transition observed for the wild‐type enzyme. The conformational and/or dynamic effects of the mutation on the H388Q enzyme are therefore various and not solely localised in the hinge region.
Purification and characterisation of bovine spleen ADPase
Authors: MOODIE Fleur D. L. , BAUM Harold , BUTTERWORTH Peter J. , PETERS Timothy J.
Published: March 2005

ADPase has been purified from bovine spleen. Electrophoresis revealed a minor contaminent in the preparation that may represent ADPase that has been decreased in size by limited proteolysis during extraction and purification. The native enzyme behaves on SDS/PAGE as a 100‐kDa protein but ADPase is a glycoprotein and so its electrophoretic behaviour may be anomalous. The apparent molecular mass is decreased to 70 kDa after removal of carbohydrate by treatment with a glycosidase. The use of a cross‐linking reagent followed by electrophoresis suggests that the enzyme is composed of a single subunit. The specific activity of the purified material was 115 U/mg protein.

The enzyme catalyses the hydrolysis of nucleoside di‐ and tri‐phosphates but nucleoside monophosphates are not acted upon. The Km for ADP (approx. 10–15 μM) is unaffected by the state of purification of the enzyme, but catalytic activity of the purified material is stimulated by inclusion of detergent in the assay system and by calcium ions. Maximum activity is seen at pH 8.0–8.5 with ADP as substrate but the optimum shifts to 7.5–8.0 for ATP hydrolysis.
Mercurial activation of human polymorphonuclear leucocyte procollagenase
Authors: BLÄSER Jörg , KNÄUPER Vera , OSTHUES Anja , REINKE Heinz , TSCHESCHE Harald
Published: March 2005

The mechanism of human polymorphonuclear leucocyte (PMNL) procollagenase activation by HgCl2 was investigated by kinetic and sequence analysis of the reaction products. HgCl2 activated PMNL procollagenase by intramolecular autoproteolytic cleavage of the Asn53–Val54 peptide bond to generate a collagenase species of Mr 65000, which was immediately converted into a second intermediate collagenase form by further autoproteolytic cleavage of the Asp64–Met65 peptide bond within the propeptide domain. This intermediate form (Met65 N‐terminus) reached maximum concentrations after 45 min and displayed only about 40% of the maximum available enzymatic activity. Final activation was obtained after autoproteolytic cleavage of either Phe79‐Met80 or Met80‐Leu81 peptide bonds. Furthermore, activation in the presence of TIMP‐1 did not suppress the intramolecular autoproteolytic cleavage of the Asn53‐Val54 peptide bond. Complete inhibition of further autoproteolytic decay of the enzyme or generated peptides was observed, which was obviously due to complex formation between the intermediate collagenase form (Val54 N‐terminus) and inhibitor, which was visualized using the Western blot technique. Thus PMNL procollagenase activation by HgCl2 followed a three‐step activation mechanism which is entirely different from the known activation mechanisms of the fibroblast matrix metalloproteinases.
Purification of two forms of arachidonate 15‐lipoxygenase from human leukocytes
Authors: IZUMI Takashi , RÅDMARK Olof , JÖRNVALL Hans , SAMUELSSON Bengt
Published: March 2005

Two different proteins with arachidonate 15‐lipoxygenase activity have been purified to near homogeneity from human leukocytes. Both have the same molecular mass (74 kDa) on SDS/PAGE and appear to be equally active with three different fatty acid substrates. The N‐terminal amino acid sequences of both forms were identical to the sequence of human reticulocyte 15‐lipoxygenase [Sigal, E., Criak, C. S., Highland, E., Grunberger, D., Costello, L. L., Dixon, R. A. F. & Nadel, J. A. (1988) Biochem. Biophys. Res. Commun. 157 457–464]. The two forms of 15‐lipoxygenase could be clearly separated by cation‐exchange chromatography. Of particular interest, the relative amounts of the two forms differed markedly between leukocytes obtained from donors and leukocyltes from an individual with eosinophilia.
Small and medium‐angle X‐ray analysis of bacterial lipoteichoic acid phase structure
Authors: LABISCHINSKI Harald , NAUMANN Dieter , FISCHER Werner
Published: March 2005

X‐ray scattering analysis was performed on various types of bacterial lipoteichoic acid in solution. The X‐ray data show that all samples investigated were characterized by a similar micellar ultrastructure (hydrophilic moiety on the outside) with a fatty acid chain conformation of the disordered, α‐type at all temperatures between 5°– 53°C. The size distribution of Staphylococcus aureus lipoteichoic acid micelles was sufficiently homogeneous to determine their size and some related molecular parameters by detailed small‐angle X‐ray scattering analysis. Nearly independent of the degree of d‐alanine substitution and the ionic strength of the aqueous dispersion, an average micelle contained about 150 lipoteichoic acid molecules arranged in a spherical assembly with a diameter of about 22 nm, whereby the hydrophilic region occupied an outer shell of about 8.5 nm thickness. Based on the average chain length of lipoteichoic acid, it could be estimated that each glycerophosphate residue contributed by about 0.34 nm to the thickness of the hydrophilic shell as compared to a theoretical value of approximately 0.8 nm for a fully extended chain conformation, indicating a highly coiled conformation of the hydrophilic chain. The bearing of these findings on the properties of membrane‐associated and secreted lipoteichoic acids is discussed.
EPR characterization of a high‐spin system in carbon monoxide dehydrogenase from Methanothrix soehngenii
Authors: JETTEN Mike S. M. , Pierik Antonio J. , HAGEN Wilfred R.
Published: March 2005

Carbon monoxide dehydrogenase and methyl‐coenzyme M reductase were purified from 61Nienriched and natural‐abundance nickel‐grown cells of the methanogenic archae Methanothrix soehngenii. The nickel‐EPR signal from cofactor F‐430 is metyl‐CoM reductase was of substoichiometric intensity and exhibited near‐axial symmetry with g= 2.153, 2.221 and resolved porphinoid nitrogen superhyperfine splittings of ∼ 1 mT. In the spectrum from 61Ni‐enriched enzyme a wellresolved parallel I= 3/2 nickel hyperfine splitting was observed, A‖= 4.4 mT. From a computer simulation of this spectrum the final enrichment in 61Ni was estimated to be 69%, while the original enrichment of the nickel metal was 87%. Carbon monoxide dehydrogenase isolated from the same batch exhibited four different EPR spectra. However, in none of these signals could any splitting or broadening from 61Ni be detected. Also, the characteristic g= 2.08 EPR signals found in some other carbon monoxide dehydrogenases and ascribed to a Ni‐Fe‐C complex, was never observed by us under any conditions of detection (4 to 100 K) and incubation in the presence of ferricyanide, dithionite, CO, coenzyme A, or acetyll‐coenzyme A. Novel, high‐spin EPR weas found in the oxidized enzyme with effective g‐values at g= 14.5, 9.6, 5.5, 4.6, 4.2, 3.8. The lines at g= 14.5 and 5.5 were tentatively ascribed to an S= 9/2 system (∼ 0.3 spins/αβ) with rhombicity E/D= 0.047 and D < 0. The other signals were assigned to an S= 5/2 system (0.1 spins/αβ) with E/D= 0.27. Both sets of signals disappear upon reduction with Em.7.5=–280 mV. With a very similar reduction potential, Em.7.5=–261 mV, an S= ‐261 mV, an S= 1/2 signal (0.1 spins/αβ) appears with the unusual g‐tensor 2.005, 1.894, 1.733. Upon further lowering of the potential the putative double cubane signal also appears. At a potential E∼ ‐320 mV the double cubane is only reduced by a few percent and this allows the detection of individual cubane EPR not subjected to dipolar interaction; a single spectral component is observed with g‐tensor 2.048, 1.943, 1.894.
Interaction of lipoamide dehydrogenase with the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: SCHULZE Egbert , BENEN Jacques A. E. , WESTPHAL Adrie H. , de KOK Arie
Published: March 2005

The interaction between lipoamide dehydrogenase (E3) and dihydrolipoyl transacetylase (E2p) from the pyruvate dehydrogenase complex was studied during the reconstitution of monomeric E3 apoenzymes from Azotobacter vinelandii and Pseudomonas fluorescens. The dimeric form of E3 is not only essential for catalysis but also for binding to the E2p core, because the apoenzymes as well as a monomeric holoenzyme from P. fluorescens, which can be stabilized as an intermediate at 0°C, do not bind to E2p.

Lipoamide dehydrogenase from A. vinelandii contains a C‐terminal extension of 15 amino acids with respect to glutathione reductase which is, in contrast to E3, presumably not part of a multienzyme complex. Furthermore, the last 10 amino acid residues of E3 are not visible in the electron density map of the crystal structure and are probably disordered. Therefore, the C‐terminal tail of E3 might be an attractive candidate for a binding region. To probe this hypothesis, a set of deletions of this part was prepared by site‐directed mutagenesis. Deletion of the last five amino acid residues did not result in significant changes. A further deletion of four amino acid residues resulted in a decrease of lipoamide activity to 5% of wild type, but the binding to E2p was unaffected. Therefore it is concluded that the C‐terminus is not directly involved in binding to the E2p core. Deletion of the last 14 amino acids produced an enzyme with a high tendency to dissociate (Kd approximately 2.5 μM). This mutant binds only weakly to E2p. The diaphorase activity was still high. This indicates, together with the decreased Km for NADH, that the structure of the monomer is not appreciably changed by the mutation. Rather the orientation of the monomers with respect to each other is changed.

It can be concluded that the binding region of E3 for E2p is constituted from structural parts of both monomers and binding occurs only when dimerization is complete.
Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN‐III‐ompA system
Authors: AUERSWALD Ennes A. , GENENGER Gabriele , MENTELE Reinhardt , LENZEN Sabine , ASSFALG‐MACHLEIDT Irmgard , MITSCHANG Lorenz , OSCHKINAT Hartmut , FRITZ Hans
Published: March 2005

A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN‐III‐ompA Escherichia coli expression system. After osmotic shock of the E. coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S‐carboxymethylpapain affinity chromatography. The resulting inhibitory material was characterized by SDS/PAGE, reversed‐phase HPLC, peptide mapping and amino acid sequencing. The recombinant variant chicken AEF‐[S1 → M, M29 → I, M89 → L]cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin. The purified variant showed a native‐chicken‐cystatin‐like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N‐labelled recombinant inhibitor were compared with those of the natural inhibitor.
Characterization of wild‐type and mutant M13 gene V proteins by means of 1H‐NMR
Authors: FOLKERS Paul J. M. , STASSEN Alphons P. M. , van DUYNHOVEN John P. M. , HARMSEN Ben J. M. , KONINGS Ruud N. H. , HILBERS Cornelis W.
Published: March 2005

Recording of good quality NMR spectra of the single‐stranded DNA binding protein gene V of the bacteriophage M13 is hindered by a specific protein aggregation effect. Conditions are described for which NMR spectra of the protein can best be recorded. The aromatic part of the spectrum has been reinvestigated by means of two‐dimensional total correlation spectroscopy. Sequence‐specific assignments were obtained for all of the aromatic amino acid residues with the help of a series of single‐site mutant proteins. The solution properties of the mutants of the aromatic amino acid residues have been fully investigated. It has been shown that, for these proteins, either none or only local changes occur compared to the wild‐type molecule. Spin‐labeled oligonucleotide‐binding studies of wild‐type and mutant gene V proteins indicate that tyrosine 26 and phenylalanine 73 are the only aromatic residues involved in binding to short stretches of single‐stranded DNA. The degree of aggregation of wild‐type gene V protein is dependent on both the total protein and salt concentration. The data obtained suggest the occurrence of specific protein—protein interactions between dimeric gene V protein molecules in which the tyrosine residue at position 41 is involved. This hypothesis is further strengthened by the observation that the solubility of tyrosine 41 mutants of gene V protein is significantly higher than that of the wild‐type protein. The discovery of the so‐called ‘solubility’ mutants of M13 gene V protein has finally made it possible to study the solution structure of gene V protein and its interaction with single‐stranded DNA by means of two‐dimensional NMR.
The endo‐1,4‐β‐glucanase I from Trichoderma reesei
Authors: BIELY Peter , VRšANSKÁ Mária , CLAEYSSENS Marc
Published: March 2005

The reaction mechanism of the non‐specific endo‐1,4‐β‐glucanase from Trichoderma reesei QM 9414 (endoglucanase I) was investigated using both reducing‐end3H‐labelled and universally 14C‐labelled cellooligo‐saccharides, as well as reducing‐end3H‐labelled xylooligosaccharides.

The bond cleavage frequencies of cellooligosaccharides proved to be dependent upon the substrate concentration, especially in the case of cellotriose. In addition to simple hydrolytic cleavage, the enzyme catalyzes reactions along alternative pathways, including transglycosylations leading to products larger than the substrate. Some of these pathways were shown to be reversible.

During cellotriose or cellopentaose degradation, substrate resynthesis was demonstrated by incorporation of added radioactive d‐glucose or cellobiose.

The endoglucanase I is active on xylan and xylooligosaccharides, but less than on soluble cellulose derivatives (e.g. hydroxyethylcellulose) and cellooligosaccharides. The fact that for these different types of substrates the same active site is operative is proven by the ability of the enzyme to utilize celloligosaccharides and xylooligosaccharides as both glycosyl donors and acceptors. The mixed substrate reactions lead to products composed of d‐glucosyl and d‐xylosyl residues.

The kinetic parameters for cellooligosaccharide degradation can be used for the description of an extended substrate binding site. Of the four putative glycosyl subsites, –II and + II show the highest affinities, 16.7 kJ · mol–1 and 7.1 kJ · mol–1, respectively.
Catabolism of platelet‐activating factor and its acyl analog
Authors: AARSMAN Anton J. , NEYS Fred W. , VAN DEN BOSCH Henk
Published: March 2005

Recent investigations have shown the presence of 1‐acyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine, i.e. the acyl analog of platelet‐activating factor (PAF), in unstimulated tissues as well as its formation along with platelet‐activating factor upon stimulation of a variety of cells. We demonstrate here that this acyl analog of PAF can be catabolized by purified lysophospholipases I and II from bovine liver with near stoichiometric formation of 2‐acetyl‐sn‐glycero‐3‐phosphocholine. Lysophospholipase II also deacetylated PAF to lysoPAF and evidence is presented to show that this is an intrinsic activity of this enzyme. This suggested that some lysophospholipases may contribute to intracellular inactivation of PAF by deacetylation. Anion‐exchange chromatography of rat liver cytosol confirmed this possibility. However, similar experiments with rat kidney cytosol and rat and human platelet cytosol clearly separated lysophospholipase activities without PAF acetylhydrolase activity from specific PAF acetylhydrolases not having lysophospholipase activity. Thus, lysophospholipases are clearly involved in the metabolism of the acyl analog of PAF and in some tissues, such as liver, may even contribute to abolishing the biological activity of PAF through deacetylation.
Structural and immunochemical studies of O‐specific polysaccharide of Proteus vulgaris 5/43 belonging to OX19 group (O‐variants)
Authors: VINOGRADOV Eugeny V. , KACA Wieslaw , RÓŻALSKI Antoni , SHASHKOV Alexander S. , CEDZYNSKI Maciej , KNIREL Yuriy A. , KOCHETKOV Nikolay K.
Published: March 2005

O‐specific polysaccharide was obtained on mild acid degradation of lipopolysaccharide of Proteus vulgaris 5/43 belonging to OX19 (O‐variants). It was found to contain d‐glucose, 2‐acetamido‐2‐deoxy‐d‐glucose, and 2‐acetamido‐2,6‐dideoxy‐l‐glucose (QuiNAc, N‐acetyl‐l‐quinovosamine) in the ratio of about 1:2:1, the last‐named sugar being rather uncommon for bacterial antigens. A computer‐assisted 13C‐NMR‐based analysis, methylation analysis, and selective cleavage with anhydrous hydrogen fluoride and dilute hydrochloric acid were applied for structural elucidation of the polysaccharide and the following structure was established: → 2)‐β‐d‐Glcp‐(1 → 6)‐α‐d‐GlcpNAc‐(1 → 3)‐α‐l‐QuipNAc‐(1 → 3)‐β‐d‐GlcpNAc‐(1 →.

Serological studies of the O‐antigen and oligosaccharides derived therefrom revealed the importance of the trisaccharide fragment β‐d‐Glcp‐(1 → 6)‐α‐d‐GlcpNAc‐(1 → 3)‐α‐l‐QuipNAc in manifesting the antigenic specificity. Serological cross‐reactions were demonstrated between lipopolysaccharides of P. vulgaris 5/43, 8/44, and OX19 strains.
Purification, primary structure and molecular cloning of a rat ribosomal protein showing homology with yeast ribosomal protein YL34
Authors: SVOBODA Michal , CICCARELLI Ernesto , VANDERMEERS‐PIRET Marie‐Claire , NAGY Anne‐Marie , VAN DE WEERDT Cécile , BOLLEN Alex , VANDERMEERS André , CHRISTOPHE Jean
Published: March 2005

A new 17‐kDa mammalian ribosomal protein (PR17) was purified to homogeneity from the rat exocrine pancreas. The purification procedure was based on acidic extraction of a heat‐denatured homogenate, ammonium‐sulfate precipitation, hydrophobic chromatography on phenyl–Sepharose and analytical reverse‐phase HPLC on μBondapak C18. Fractions of interest were collected using an antiserum directed against the first (1–14) moiety of somatostatin(1–28). 30 μg pure RP17 were obtained from 1 g fresh pancreas.

A short 111‐b cDNA encoding RP17 was amplified from rat pancreatic first‐strand cDNA template by using two 64‐fold degenerate heptadecamer primers in the DNA‐polymerase‐chain reaction. From the sequence of amplified cDNA, an unambiguous oligonucleotide probe was designed to screen a rat pancreatic cDNA library. A cDNA clone coding for RP17 was isolated, whose nucleotide sequence, with an open reading frame coding for 155 amino acids (molecular mass of 17199 Da), confirmed the partial amino acid sequences directly obtained from the purified protein.

Northern‐blot analysis showed that a similar 0.75‐kb transcript was present in rat pancreas, in the rat pancreatic acinar cell line AR 4‐2J and in the human neuroblastoma cell‐line NB‐OK‐1, the highest level being in the latter two preparations, despite similar levels of RP17 in all three preparations, as tested with a rabbit antiserum directed against purified RP17.

The N‐terminal sequence of both RP17 and the ribosomal protein YL43 from Saccharomyces cerevisiae (39 amino acid residues) showed a high degree of identity (77%), indicating that RP17 is a mammalian homolog of yeast ribosomal protein YL43.
Interaction of the low‐molecular‐mass, guanine‐nucleotide‐binding protein with the actin‐binding protein and its modulation by the cAMP‐dependent protein kinase in bovine platelets
Authors: UEDA Masahiro , OHO Chikara , TAKISAWA Haruhiko , OGIHARA Satoshi
Published: March 2005

Platelets have been shown to possess several, different, low‐molecular‐mass, guanine‐nucleotide‐binding proteins (G‐proteins) with molecular masses about 20–30 kDa. We report here that a 25‐kDa G‐protein copurified with the bovine platelet actin‐binding protein (ABP), a cross‐linker of actin filaments which is known to generate the three‐dimensional network of actin. Both the G‐protein and ABP were recovered in a fraction that was insoluble in Triton X‐100 and were extracted in 0.6 M NaCl. Gel‐filtration chromatography of the high‐salt extract and rechromatography in a low‐salt solution indicated that the two proteins may be associated with each other. The association of the two proteins was suggested by cosedimentation of the G‐protein with the actin gel formed by actin and ABP. The amounts of the cosedimented G‐protein and ABP was unaffected by guanosine‐5′‐O‐[β‐thio]diphosphate and guanosine‐5′‐O‐[γ‐thio]triphosphate, but the G‐protein, not ABP, was partially released from the actin gel by phosphorylating ABP with cAMP‐dependent protein kinase. Thus, the association of the two proteins was affected by modification of ABP, but not by modification of G‐proteins. The physiological significance of the possible association of the two proteins might be that the membrane skeleton functions as a modulator of the G‐protein, rather than that the G‐protein modulates the function of the membrane skeleton which comprises ABP.
Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin‐NADP+ reductase
Authors: MEDINA Milagros , PELEATO M. Luisa , MENDEZ Enrique , GOMEZ‐MORENO Carlos
Published: March 2005

Flavodoxin from the nitrogen‐fixing cyanobacteria Anabaena PCC 7119 forms an electron‐transfer complex with ferredoxin – NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side‐chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water‐soluble carbodiimide, N‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time‐dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH – cytochrome‐c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non‐covalent complex with FNR was fourfold higher.

After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1‐14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long‐chain flavodoxins show an insert compared to short‐chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three‐dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long‐chain flavodoxins, suggesting that they could play an essential role in substrate recognition.