bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and properties of aminopeptidase H from chicken skeletal muscle
Authors: RHYU Mee Ra , NISHIMURA Toshihide , KATO Yutaka , OKITANI Akihiro , KATO Hiromichi
Published: March 2005

Aminopeptidase H was purified from fresh chicken breast muscle by ammonium sulfate fractionation and successive chromatographies on DEAE‐cellulose, Ultrogel AcA 34, activated thiol‐Sepharose 4B, phenyl‐Sepharose CL‐4B and DEAE‐cellulose again. The purified enzyme migrated as a single band on SDS/PAGE. Aminopeptidase H exhibits activity against both L‐leucine β‐naphthylamide and α‐N‐benzoyl‐DL‐arginine β‐naphthylamide. The molecular mass of this enzyme was found to be 52 kDa on SDS/PAGE and 400 kDa on Sepharose 6B column chromatography. The optimum pH for the hydrolysis of both substrates was 8.0 and this activity was remarkably enhanced by reducing agents. The enzyme was strongly inhibited by monoiodoacetate and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, bestatin or puromycin. Aminopeptidase H has been shown to hydrolyze di‐, tri‐ and tetrapeptides in the manner of an aminopeptidase, as well as the β‐naphthylamide derivatives of amino acids. However, the enzyme has not been shown to hydrolyze proteins such as hemoglobin, bovine serum albumin, myofibrillar proteins or sarcoplasmic proteins.
Thermodynamics of cation binding to Nereis sarcoplasmic calcium‐binding protein
Authors: LUAN‐RILLIET Ying , MILOS Mladen , COX Jos A.
Published: March 2005

Nereis sarcoplasmic calcium‐binding protein contains three functional EF‐hand sites which bind Ca2+ or Mg2+ competitively. Here it was confirmed over a large range of [Mg2+] that the positive cooperativity in binding of Ca2+ (nH= 2) is the result of allostery in Mg2+ dissociation. At pH 7.5, Ca2+ or Mg2+ binding provokes the release of 1.4 mol proton/mol protein, whereas no protons are released during Ca2+‐Mg2+ exchange. The enthalpy change as a function of Ca2+ binding yields a two‐step curve with an inflection point at 1 mol Ca2+/mol protein and a maximum of –66 kJ/mol at 3 mol Ca2+/mol protein. Binding of three Mg2+ ions is cooperative (nH= 1.8) with a maximal enthalpy change of –15.1 kJ/mol protein. Difference spectroscopy led to the conclusion that, in the metal‐free protein, the structure around the aromatic residues is well organized, but that Tyr and Trp residues are still solvent‐exposed. Upon Ca2+ binding Tyr and Trp spectra are blue‐shifted, but some Trp residues are confined to a positively charged pocket. Examination of the Ca2+‐saturated three‐dimensional crystal structure confirmed that Trp4 and Trp57 are located in such pockets or clefts, close to the surface. During the allosteric T → R transition, promoted by binding of the first Mg2+, the Trp residues move to a hydrophobic environment. For both Ca2+ and Mg2+, the enthalpy change and the conformational change in the environment of the aromatic residues is much more pronounced in the first, than in the subsequent two binding steps. In this respect, the latter seem to be equivalent.
Kinetic behaviour of α‐chymotrypsin in reverse micelles
Authors: MAO Qingcheng , WALDE Peter , LUISI Pier Luigi
Published: March 2005

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of α‐chymotrypsin have been studied in reverse micelles formed by sodium bis(2‐ethylhexyl)sulfosuccinate in isooctane. Pre‐steady state and steady‐state kinetic constants, in water and in reverse micelles, have been determined by stopped‐flow spectrophotometry for the hydrolysis of two substrates, namely acetyl‐L‐tryptophan‐p‐nitrophenyl ester and p‐nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.
Modulation of cell‐surface transferrin receptor by the imino sugar N‐butyldeoxynojirimycin
Authors: PLATT Frances M. , KARLSSON Gunilla B. , JACOB Gary S.
Published: March 2005

The imino sugar, N‐butyldeoxynojirimycin, is an inhibitor of the glycoprotein‐processing enzyme glucosidase I and exhibits anti‐(human immunodeficiency virus) activity in vitro. We have investigated the effect(s) of this compound on cell‐surface glycoproteins by flow cytometry. We observed selective modulation of the transferrin receptor in response to treatment with 0.5 mM N‐butyldeoxynojirimycin resulting in reduced cell‐surface transferrin‐receptor expression. The receptor modulation was dose dependent, resulted in reduced 59Fe uptake by treated cells and was fully reversible within 24 h of culture in the absence of the compound.

Pulse/chase analysis in conjunction with endoglycosidase‐H digestion demonstrated that transferrin‐receptor glycosylation was altered following N‐butyldeoxynojirimycin treatment, which is compatible with glucosidase inhibition. In addition, modulation of transferrin receptor in response to N‐butyldeoxynojirimycin was not confined to a single cell line, but was also observed with certain human lymphoid and myeloid cell lines. Mechanism(s) of action of the imino sugar resulting in reduced cell‐surface transferrin‐receptor expression are discussed.
Intermediates of peroxisomal β‐oxidation of [U‐14C]hexadecanedionoate
Authors: POURFARZAM Morteza , BARTLETT Kim
Published: March 2005

1. The oxidation of [U‐14C]hexadecanedionoyl‐mono‐CoA was stimulated by CoA, by carnitine in the absence of CoA and by the presence of an NAD+‐regenerating system.

2. Substrate inhibition was observed with respect to [U‐14C]hexadecanedionoyl‐mono‐CoA at concentrations greater than 35 μM.

3. Acetyl‐CoA and the dicarboxyl‐CoA esters of chain length C6–16 were detected by HPLC under standard incubation conditions.

4. In the absence of the NAD+‐regenerating system, 2‐enoyl‐CoA and 3‐hydroxyacyl‐CoA esters were detected.

5. In general, the peroxisomal β‐oxidation of dicarboxylates is very similar to that of monocarboxylates [Bartlett, K., Hovik, R., Eaton, S., Watmough, N.J. & Osmundsen, H. (1990) Biochem. J. 270, 175–180] except that chain shortening does not proceed beyond C6.

6. We conclude that the peroxisomal β‐oxidation of dicarboxylates is regulated by the redox state of the peroxisomal matrix and CoA availability.
Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome‐c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron‐sulfur subunit
Authors: GEIER Birgitta M. , SCHÄGGER Hermann , BRANDT Ulrich , COLSON Anne‐Marie , VON JAGOW Gebhard
Published: March 2005

Cytochrome‐c reductase was isolated from Saccharomyces cerevisiae GM50‐3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE.

Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome‐c reductase, were analysed. The single amino acid substitution in the cytochrome‐b subunit, N256Y in the mutant Myx‐119 and G137R in the mutant Myx‐118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations.

The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher.

Cytochrome‐c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate‐stilbene and myxothiazol by means of fluorescence‐quench and red‐shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron‐sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type.

This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron‐sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron‐sulfur subunit.
A soluble form of the glycolipid‐anchored receptor for urokinase‐type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria
Authors: PLOUG Michael , ERIKSEN Jens , PLESNER Torben , HANSEN Niels Ebbe , DANØ Keld
Published: March 2005

The cellular urokinase‐type plasminogen‐activator (uPA) receptor (uPAR) is a glycolipid‐anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand‐binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol‐specific phospholipase C (PLC) or endoproteinase Asp‐N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem‐cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA‐binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH‐affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR‐specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns‐specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C‐terminus close to the proposed glycolipid‐attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as‐yet‐unidentified trans‐amidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.
Molecular cloning of a new bombesin receptor subtype expressed in uterus during pregnancy
Authors: GORBULEV Valentin , AKHUNDOVA Aida , BÜCHNER Hubert , FAHRENHOLZ Falk
Published: March 2005

The homology screening approach has been used to clone a new member of the guanine‐nucleotide‐binding‐protein‐coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399‐amino‐acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin‐releasing‐peptide (GRP) receptor and the neuromedin‐B receptor, respectively. Binding experiments with the stably transfected LLC‐PK1 cell line expressing the new receptor protein confirmed the bombesin‐like nature of the cloned receptor. The relative order of ligand affinity, GRP = neuromedin C ≫ neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin‐B subtype of bombesin receptors. Northern‐blot analysis of mRNA species from several guinea‐pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals.
Effect of substrate structure on the activity of Man9‐mannosidase from pig liver involved in N‐linked oligosaccharide processing
Authors: BAUSE Ernst , BREUER Wilhelm , SCHWEDEN Jürgen , ROESER Rainer , GEYER Rudolf
Published: March 2005

Man9‐mannosidase, an α1,2‐specific enzyme located in the endoplasmic reticulum and involved in N‐linked‐oligosaccharide processing, has been isolated from crude pig‐liver microsomes and its substrate specificity studied using a variety of free and peptide‐bound high‐mannose oligosaccharide derivatives. The purified enzyme displays no activity towards synthetic α‐mannosides, but removes three α1,2‐mannose residues from the natural Man9‐(GlcNAc)2 substrate (M9). The α1,2‐mannosidic linkage remaining in the M6 intermediate is cleaved about 40‐fold more slowly. Similar kinetics of hydrolysis were determined with Man9‐(GlcNAc)2 N‐glycosidically attached to the hexapeptide Tyr‐Asn‐Lys‐Thr‐Ser‐Val (GP‐M9), indicating that the specificity of the enzyme is not influenced by the peptide moiety of the substrate. The α1,2‐mannose residue which is largely resistant to hydrolysis, was found to be attached in both the M6 and GP‐M6 intermediate to the α1,3‐mannose of the peripheral α1,3/α1,6‐branch of the glycan chain. Studies with glycopeptides varying in the size and branching pattern of the sugar chains, revealed that the relative rates at which the various α1,2‐mannosidic linkages were cleaved, differed depending on their structural complexity. This suggests that distinct sugar residues in the aglycon moiety may be functional in substrate recognition and binding. Reduction or removal of the terminal GlcNAc residue of the chitobiose unit in M9 increased the hydrolytic susceptibility of the fourth (previously resistant) α1,2‐mannosidic linkage significantly. We conclude from this observation that, in addition to peripheral mannose residues, the intact chitobiose core represents a structural element affecting Man9‐mannosidase specificity. A possible biological role of the enzyme during N‐linked‐oligosaccharide processing is discussed.
Activation by ATP of a proton‐conducting pathway in yeast mitochondria
Authors: PRIETO Susana , BOUILLAUD Frédéric , RICQUIER Daniel , RIAL Eduardo
Published: March 2005

The growth of Saccharomyces cerevisiae cells under aerobic conditions, in the presence of an energy‐rich source, leads to production of an excess of NAD(P)H. Since the redox balance must be maintained, it has been postulated that NAD(P)H reoxidation is accelerated by the activation of energy‐dissipating reactions, which would, in turn, explain the low growth efficiencies observed. It has been demonstrated already in S. cerevisiae cultures that these putative energy‐dissipating reactions are stimulated both by oxygen and high cytosolic ATP levels. In this paper, we show that ATP induces a proton‐permeability pathway in mitochondria at concentrations which are within the physiological range, as revealed both from the ATP stimulation of respiration and from the induction of H+ ‐dependent swelling. We also demonstrate that phosphate acts as a competitive inhibitor of the nucleotide, and since activation is observed even in the presence of atractylate, we postulate that the ATP‐binding site is located in the outer face of the mitochondrial inner membrane.
Transiently and stably introduced CCAAT/enhancer‐binding‐protein genes are constitutively expressed in cultured cells
Authors: XANTHOPOULOS Kleanthis G. , CANNON Paul D. , ROBINSON Gregory S. , MIRKOVITCH Jovan , DARNELL James E.
Published: March 2005

CCAAT/enhancer‐binding protein (C/EBP) is expressed in certain cell types including hepatocytes and adipocytes. In order to understand the mechanisms that control the expression of the mouse C/EBP gene in the liver as well as in adipocytes, we have studied both the endogenous gene and transfected C/EBP gene constructs. The initiation site of transcription was identified and a strong liver‐specific DNase‐1 hypersensitive site located at ‐ 3 kb, which does not appear to contribute functionally to the regulation of the gene in a variety of either transiently or stably transfected cells with constructs which include sequences up to 6‐kb upstream of the transcription start. C/EBP gene expression during the transition from preadipocytes to adipocytes was shown to be controlled at the level of transcription. However, adipocytes stably transfected with constructs that include – 3.3 kb upstream of the C/EBP gene do not express the reporter genes in a differentiation‐specific manner. We detected several DNA‐binding proteins that interact with the upstream sites of the C/EBP gene. Those include two labile and two heat‐stable site‐specific DNA‐binding proteins that are present in nuclear extracts from several tissues and cultured cell lines.
Ring‐substituted diaqua(1,2‐diphenylethylenediamine)platinum(II) sulfate reacts with DNA through a dissociable complex
Authors: BERNGES Frithjof , HOLLER Eggehard
Published: March 2005

Ring‐substituted diaqua(1,2‐diphenylethylenediamine)platinum(II) sulfate shows unusual kinetics in its reaction with salmon testis DNA. The mechanism for diaqua[meso‐1,2‐bis(2,6‐dichloro‐4‐hydroxyphenyl)ethylenediamine]platinum(II) sulfate, [Pt(H2O)2(meso‐6)]2+SO2−4, a representative of this series, has been investigated and compared with that for cis‐[Pt(NH3)2(H2O)2]2+. Reactions were followed by atomic absorption, analytical HPLC of Pt‐DNA digests, arrest of enzymatic DNA synthesis/degradation, ultraviolet and fluorescence spectrophotometry. Except for the formation of monofunctional DNA adducts, the kinetics of the platinum(II) complexes are comparable. The pseudo‐first‐order rate constant for the attack of DNA by [Pt(H2O)2(meso‐6)]2+ follows the concentration of DNA in a hyperbolic fashion, which is in contrast to the linear dependence for cis‐[Pt(NH3)2(H2O)2]2+. The hyperbolic dependence is typical for a dissociable DNA/drug complex preceding the coordination reaction. By studying the binding of free ligand to DNA, and by correlating ligand structures and electrostatic charges with effects on adduct formation, both the phenyl residues and the positive charge of the platinum(II) complex are shown to be crucial for the stability of the dissociable complex. A non‐intercalative mode of binding to the DNA backbone is suggested. At the high concentrations of DNA found in cell nuclei, the reaction of the dissociable complex can, principally, become rate‐limiting in the attack of DNA and thus reduce the cytotoxic efficiency of a drug.
Mechanism of the interaction of EcoRII restriction endonuclease with two recognition sites
Authors: PETRAUSKENE Olga V. , KUBAREVA Elena A. , GROMOVA Elisaveta S. , SHABAROVA Zoe A.
Published: March 2005

The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 215 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single‐site substrates by EcoRII is observed in the presence of 11–14‐bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different efficiences or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single‐site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate.
Degradation of gangliosides by the lysosomal sialidase requires an activator protein
Authors: FINGERHUT Ralph , VAN DER HORST Gijsbertus T. J. , VERHEIJEN Frans W. , CONZELMANN Ernst
Published: March 2005

Lysosomal sialidase, which was formerly believed to degrade only water‐soluble substrates but not glycolipids, cleaves ganglioside substrates II3NeuNAc‐LacCer, IV3NeuNAc, II3NeuNAc‐GgOse4Cer, IV3 NeuNAc, II3(NeuNAc)2‐GgOse4Cer when these are dispersed either with an appropriate detergent (taurodeoxycholate) or with the sulfatide activator protein, a physiologic lipid solubilizer required for the lysosomal hydrolysis of other glycolipids by water‐soluble hydrolases. In the presence of the activator protein, time and protein dependence were linear within wide limits, while the detergent rapidly inactivated the enzyme.

The disialo group of the b‐series gangliosides was only poorly attacked by the enzyme when the lipids were dispersed with the activator protein, whereas in the presence of the detergent, they were hydrolyzed as fast as terminal sialic acid residues.

With the appropriate assay method, significant ganglioside sialidase activity could be demonstrated in the secondary lysosome fraction of normal skin fibroblasts but not of sialidosis fibroblasts.

Our results support the notion that there is only one lysosomal sialidase, which degrades both the water‐soluble and the membrane‐bound sialyl glycoconjugates.
Purification and characterization of vanillyl‐alcohol oxidase from Penicillium simplicissimum
Authors: DE JONG Ed , VAN BERKEL Willem J. H. , VAN DER ZWAN Rick P. , DE BONT Jan A. M.
Published: March 2005

Vanillyl‐alcohol oxidase was purified 32‐fold from Penicillium simplicissimum, grown on veratryl alcohol as its sole source of carbon and energy. SDS/PAGE of the purified enzyme reveals a single fluorescent band of 65 kDa. Gel filtration and sedimentation‐velocity experiments indicate that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8α‐(N3‐histidyl)‐FAD from pH‐dependent fluorescence quenching (pKa= 4.85) and no decrease in fluorescence upon reduction with sodium borohydride.

The enzyme shows a narrow substrate specificity, only vanillyl alcohol and 4‐hydroxybenzyl alcohol are substrates for the enzyme. Cinnamyl alcohol is a strong competitive inhibitor of vanillylalcohol oxidation.

The visible absorption spectrum of the oxidized enzyme shows maxima at 354 nm and 439 nm, and shoulders at 370, 417 and 461 nm. Under anaerobic conditions, the enzyme is easily reduced by vanillyl alcohol to the two‐electron reduced form. Upon mixing with air, rapid reoxidation of the flavin occurs. Both with dithionite reduction and photoreduction in the presence of EDTA and 5‐deazaflavin the red semiquinone flavin radical is transiently stabilized. Opposite to most flavoprotein oxidases, vanillyl‐alcohol oxidase does not form a flavin N5‐sulfite adduct. Photoreduction of the enzyme in the presence of the competitive inhibitor cinnamyl alcohol gives rise to a complete, irreversible bleaching of the flavin spectrum.
Induction of the three peroxisomal β‐oxidation enzymes is synergistically regulated by dexamethasone and fatty acids, and counteracted by insulin in Morris 7800C1 hepatoma cells in culture
Authors: SØRENSEN Hilde Nebb , GAUTIK Kaare M. , BREMER Jon , SPYDEVOLD Øystein
Published: March 2005

This work describes the molecular mechanism of hormonal modulation of fatty‐acid peroxisomal β oxidation in liver. Morris 7800C1 hepatoma cells and isolated hepatocytes were cultured in the presence of myristic acid (1mM) and tetradecylthioacetic acid, a 3‐thia fatty acid (50 μM), separately or in combination with dexamethasone (0.25 μM) or insulin (0.4 μM).

Myristic acid stimulated acyl‐CoA oxidase and a synergistic action was observed with dexamethasone. Parallel changes were recognized in enzyme protein and mRNA levels as quantified from immunoblots and Northern analyses. Myristic acid and tetradecylthioacetic acid had similar effects on this enzyme, while insulin inhibited the basal activity and blocked all inductions by the fatty acids and dexamethasone.

Parallel mRNA and immunoblot analyses of the subsequent enzymes in the peroxisomal β‐oxidation pathway, enoyl‐CoA hydratase/3‐hydroxyacyl‐CoA dehydrogenase/Δ3,Δ2‐enoyl‐CoA isomerase and 3‐oxoacyl‐CoA thiolase, showed an even stronger induction by tetradecylthioacetic acid and dexamethasone, while the counteraction by insulin was maintained in both 7800C1 hepatoma cells and hepatocytes. In hepatoma cells, the thiolase always showed the most pronounced induction (about 40‐fold) after 14 days, with parallel changes in protein and mRNA levels.

The results suggest that the changes in peroxisomal β‐oxidation enzymes in 7800C1 hepatoma cells are due to a major effect on steady‐state mRNA levels giving rise to corresponding alterations in enzyme protein. These results may be explained by regulation at the level of transcription of corresponding genes, but mRNA stability changes and/or translational effects may also be of importance.
Characterization of small‐molecular‐mass guanine‐nucleotide‐binding regulatory proteins in insulin‐secreting cells and PC12 cells
Authors: REGAZZI Romano , VALLAR Lucia , ULLRICH Susanne , RAVAZZOLA Mariella , KIKUCHI Akira , TAKAI Yoshimi , WOLLHEIM Claes B.
Published: March 2005

The distribution of ras‐related small‐molecular‐mass guanine‐nucleotide‐binding regulatory proteins (SMG) of two insulin‐secreting cell lines, RINm5F and HIT‐T15, and of a catecholaminesecreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two‐dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin‐secreting cells, rho protein(s) that cannot be detected with the GTP‐binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin‐secreting cell line RINm5F and the catecholamine‐secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT‐T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion.
Characterization of the chicken α1(VI) collagen promoter
Authors: KOLLER Erich , TRUEB Beat
Published: March 2005

The promoter of the chicken α1(VI) collagen gene resembles the 5′‐flanking regions of many housekeeping genes. It lacks a canonical TATAA box but contains potential binding sites for transcription factors AP1 and SP1. The promoter region has a relatively high GC content and forms a typical CpG island. In accordance with the absence of a TATAA element, the gene contains multiple transcription‐initiation sites distributed over 80 bp genomic DNA. A 621‐bp fragment derived from the 5′ end of the α1(VI) collagen gene is able to direct transcription of a heterologous reporter gene in transient‐expression assays. Other DNA fragments that are either shorter or longer than the 621‐bp fragment show markedly reduced promoter activity. Thus, the basic promoter element of the α1(VI) collagen gene must reside within this 621‐bp fragment.
Cloning and overexpression of Lactobacillus helveticusd‐lactate dehydrogenase gene in Escherichia coli
Authors: KOCHHAR Sunil , HOTTINGER Herbert , CHUARD Nathalie , TAYLOR Paul G. , ATKINSON Tony , SCAWEN Michael D. , NICHOLLS David J.
Published: March 2005

NAD+‐dependent d‐lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N‐terminal sequence and amino acid residues 220–215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong‐Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499–8513], a 0.6‐kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the d‐lactate dehydrogenase gene. Both plasmids overexpressed d‐lactate dehydrogenase (> 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticusd‐lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of d‐2‐hydroxy‐acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong‐Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60–66]. The physicochemical and catalytic properties of recombinant d‐lactate dehydrogenase were identical to those of the wild‐type enzyme, e.g. α2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2‐oxo‐acid substrates. The kinetic profiles of 2‐oxo‐acid substrates showed some marked differences from that of l‐lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.
Antagonistic properties are shifted back to agonistic properties by further N‐terminal shortening of pituitary adenylate‐cyclase‐activating peptides in human neuroblastoma NB‐OK‐1 cell membranes
Authors: VANDERMEERS André , VANDENBORRE Stéphane , HOU Xue , DE NEEF Philippe , ROBBERECHT Patrick , VANDERMEERS‐PIRET Marie‐Claire , CHRISTOPHE Jean
Published: March 2005

N‐terminally shortened analogs of the 27‐amino‐acid and 38‐amino‐acid forms of the pituitaryadenylate‐cyclase‐activating neuropeptide, PACAP(1–27) and PACAP(1–38), were synthesized by a solid‐phase method. Systematic deletion of the first 13 amino acids of both PACAP was tested by evaluating their ability to occupy the specific and selective PACAP receptor of human neuroblastoma NB‐OK‐1 cell membranes and to stimulate adenylate cyclase or, when inactive per se, to inhibit PACAP‐stimulated adenylate cyclase activity. For each peptide, the Kact (concentration required for half‐maximal adenylate cyclase activation) or Ki [concentration required to shift the dose/response curve of PACAP(1–27) twofold to the right] was in good agreement with the corresponding IC50 [concentration inhibiting 50% of 125I‐[AcHis1]PACAP(1–27) binding to membranes], suggesting interaction with the same homogeneous class of adenylate cyclase‐coupled receptors.

The deletion of the two first amino acids (His1 and Ser2) sufficed to decrease the affinity for receptors and to suppress the capacity to activate adenylate cyclase. The shorter fragments 3–27 and 3–38, 4–27 and 4–38, 5–27 and 5–38, 6–27 and 6–38, 7–27 and 7–38, 8–27 and 8–38, and 9–27 and 9–38 were all competitive antagonists of PACAP(1–27)‐stimulated activity with the N‐terminally shortened PACAP(1–38) derivatives being 4–30‐fold more potent than the equivalent PACAP(1–27) derivatives. In this group PACAP(6–38) was the most potent antagonist (Ki 1.5 nM).

Surprisingly, the N‐terminally shorter fragments 10–27 and 10–38, 11–27 and 11–38, 12–27 and 12–38, 13–27 and 13–38, and 14–27 and 14–38 were again able to stimulate adenylate cyclase, the smallest fragments, PACAP(14–27) and PACAP(14–38), being the most potent and efficient (Kact 2 μM and 0.1 μM, respectively). In this group of agonists, PACAP(1–38) derivatives deleted at the N‐terminus were also more potent than the equivalent PACAP(1–27) derivatives.
Molecular cloning of the antibacterial protein of the giant African snail, Achatina fulica Férussac
Authors: OBARA Kuniaki , OTSUKA‐FUCHINO Hisako , SATTAYASAI Nison , NONOMURA Yoshiaki , TSUCHIYA Takahide , TAMIYA Toru
Published: March 2005

An expression cDNA library was constructed with poly(A)‐rich RNA extracted from the collar of the giant African snail, Achatina fulica Férussac. A 1.9‐kbp cDNA clone encoding a precursor of antibacterial glycoprotein of the snail, achacin, was isolated from the cDNA expression library. The cDNA sequence contains an open reading frame with 1593‐nucleotide residues. The deduced amino acid sequence of this achacin precursor starts with a 29‐residue leader peptide followed by a 502‐residue mature peptide (56 kDa) with four possible N‐glycosylation sites, Asn‐Xaa‐Ser or Asn‐Xaa‐Thr. The Northern‐blot analysis proved that the achacin precursor was specifically expressed in the tissue of snail collar and processed to mature achacin. cDNA inserts encoding achacin precursor were subcloned into expression plasmids. Three kinds of expressed polypeptides were cross‐reacted with rabbit antiserum raised against achacin. The largest polypeptide (Mr 63000) should be the achacin precursor.
Relationship between the major protein kinase C substrates acidic 80‐kDa protein‐kinase‐C substrate (80K) and myristoylated alanine‐rich C‐kinase substrate (MARCKS)
Authors: HERGET Thomas , BROOKS Susan F. , BROAD Simon , ROZENGURT Enrique
Published: March 2005

Two major protein‐kinase‐C (PKC) substrates have been described in the literature; an 87‐kDa bovine and human PKC substrate, called MARCKS, and an acidic 80‐kDa PKC substrate, isolated from rat brain and Swiss 3T3 cells, termed 80K. Since there is only 66–74% sequence similarity between MARCKS and 80K, we have further investigated their relationship in this study.

Southern‐blot experiments with gene‐specific probes demonstrated the presence of the 80K, but not MARCKS, gene in the mouse genome. Furthermore, polymerase‐chain‐reaction (PCR) analyses using three pairs of primers that specifically recognise either 80K, MARCKS or conserved sequences of both genes, revealed the presence of only the 80K gene in the mouse and rat genomes and only the MARCKS gene in the bovine and human genomes with mRNA expression in the corresponding brain tissues. Northern‐blot analysis of a variety of tissues indicated that both 80K and MARCKS have similar patterns of expression. Most components of signal‐transduction pathways are present in multiple molecular isoforms as members of a gene family. In contrast, the findings presented in this study indicate that rodent 80K and bovine and human MARCKS are not distinct members of a gene family, but represent the equivalent substrates in different species.
Isolation, sequence analysis and characterization of cDNA clones coding for the C chain of mouse C1q
Authors: PETRY Franz , REID Kenneth B. M. , LOOS Michael
Published: March 2005

A mouse macrophage λgt11 cDNA library was screened using a genomic DNA clone coding for the C‐chain gene of human C1q. Approximately 600000 recombinant phage plaques were hybridized with peroxidase‐labeled human C‐chain probe and detected by enhanced chemiluminescence. Five positive clones were obtained. The size of the full‐length cDNA is 1019 bp. The sequence identity of the nucleotide sequence with human C1q C chain is 79%, the identity of the deduced amino acid sequences is 73%. The mouse C1q C chain exhibits the same structural features as the human C chain, e.g. conservation of the cysteine residues. Like the mouse A chain, the mouse C chain has an RGD sequence that may be recognized by receptors of the integrin family. No RGD sequences have been found in any of the human C1q chains. The size of the C‐chain mRNA (1.2 kb) and its tissue distribution (macrophages being the cell type with the highest mRNA concentration) are identical to the mRNA of the mouse A and B chains. Alignment of human and mouse C1q A, B and C chains exhibits two blocks of highly conserved residues within the C‐terminal globular regions. Three other proteins, collagen type VIII and type X and precerebellin share this similarity with C1q, indicating the structural and probably functional importance of these regions within the non‐collagenous domains of the molecules.
Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3‐hydroxybutyric acid) in Chromatium vinosum strain D
Authors: LIEBERGESELL Matthias , STEINBÜCHEL Alexander
Published: March 2005

From a genomic library of Chromatium vinosum strain D in λL47, a 16.5‐kbp EcoRI‐restriction fragment was identified by hybridization with a DNA fragment harboring the operon for Alcaligenes eutrophus poly(3‐hydroxyalkanoate) (PHA) synthesis. This fragment and subfragments thereof restored the ability to synthesize and accumulate PHA in PHA‐negative mutants of A. eutrophus. A region of 6977 bp was sequenced; seven open reading frames (ORFs) were identified which probably represent coding regions; six of these are most probably relevant for PHA biosynthesis in C. vinosum. The structural genes for biosynthetic acetyl‐CoA acyltransferase (β‐ketothiolase; phbACv, 1188 bp) and NADH‐dependent acetoacetyl‐CoA reductase (phbBCv, 741 bp) were separated by ORF4 (462 bp) and ORF5 (369 bp). Downstream of phbBCv ORF7 (471 pb) was identified which was not completed at the 3′ terminus. The functions of ORF4, ORF5, and ORF7 are not known. The amino acid sequences of β‐ketothiolase and acetoacetyl‐CoA reductase deduced from phbACv and phbBCv, exhibited a similarity of 68.2% and 56.4% identical amino acids, respectively, to the corresponding enzymes of A. eutrophus. Antilinear to and upstream of the genes mentioned above, two genes were identified which were transcribed from a σ70‐dependent promoter. This promoter overlapped with and was divergent to the phbACv promoter; the transcriptional start sites were mapped by S1 nuclease protection assays. These genes were ORF2 (1074 bp), whose function is not known but whose presence in Escherichia coli is essential for expression of PHA synthase activity, and the structural gene for a PHA synthase of low Mr (phbCCv, 1068 bp). The gene products of ORF2 and phbCCv, with Mr of 40525 and 39730, respectively, were expressed in E. coli applying the T7 RNA polymerase/promoter system. Although the amino acid sequence of PHA synthase deduced from phbCCv exhibited only 24.7% overall similarity with the PHA synthase of A. eutrophus, highly conserved regions were identified.
Efficiency of the 5′‐terminal sequence (Ω) of tobacco mosaic virus RNA for the initiation of eukaryotic gene translation in Escherichia coli
Authors: IVANOV Ivan G. , ALEXANDROVA Roumjana , DRAGULEV Bojan , LECLERC Denis , SARAFFOVA Adriana , MAXIMOVA Vera , ABOUHAIDAR Mounir G.
Published: March 2005

Recent studies have demonstrated that the 5′ leader (Ω sequence) of tobacco mosaic virus RNA has a certain enhancing capacity for translation of mRNA in both prokaryotes and eukaryotes. In order to estimate the efficiency of Ω to initiate translation of mRNA in Escherichia coli, in comparison to the Shine‐Dalgarno (S/D) sequence, we have inserted eight different eukaryotic genes into two types of E. coli expression vectors containing one constitutive promoter (P1) but different translation‐initiation sites (S/D or ΩΔ3 sequence, respectively). The efficiency of transcription and translation in vivo was evaluated for these vectors by measuring the yield of protein and both the level and stability of mRNA. We report that substitution of ΩΔ3 for S/D decreases the yield of expressed protein 4–1900‐fold and the content of gene‐specific mRNA is decreased by about sevenfold. However, in comparison with the S/D sequence, the level of protein expressed under the translational control of ΩΔ3 is less sensitive to changes in the 5′ coding region. We also report that the Ω sequence contains a region of 10–12 nucleotides complementary to the small ribosomal subunit RNA (rRNA) of E. coli, Eikenella corrodens and Xenopus laevis, and to the rRNA of the (small ribosomal) subunit of Oryza sativa.
The structure of the mammalian antibacterial peptide cecropin P1 in solution, determined by proton‐NMR
Authors: SIPOS David , ANDERSSON Mats , EHRENBERG Anders
Published: March 2005

Cecropins are peptides with antibacterial activity originally found in insects. Recently a cecropin‐type peptide was isolated from pig intestine. This peptide, porcine cecropin P1, which has 31 amino acid residues and is not amidated in the C‐terminus, has been synthesized, purified, and investigated by CD and two‐dimensional 1H‐NMR at pH 5.0 in aqueous solution with 30% (by vol.) 1,1,1,3,3,3‐hexafluoro‐2‐propanol. All proton resonances have been assigned except for the N‐terminal serine. Using constraints derived from NOE connectivities and 3JNHα‐coupling constants, three‐dimensional structures have been calculated by means of a distance‐geometry program. Some of these structures have been refined by energy minimization and restrained molecular dynamics. The structures reveal an α‐helix of approximately seven turns along nearly the full length of the peptide. The central part of the helix is very well defined by the NMR constraints. Also the chemical shifts of the α protons and the results of CD measurements are in accord with this structure, which is different from the helix‐hinge‐helix structure earlier found in cecropin A and related peptides. In the α‐helix of cecropin P1 there is a long amphipathic section, of 4–5 turns, and a short hydrophobic section of one to two turns, with an intervening Glu‐Gly sequence, which is a potential bend‐forming section. The helix can easily span a lipid membrane.
Short‐chain dehydrogenases
Authors: KROOK Maria , PROZOROVSKI Vladimir , ATRIAN Silvia , GONZÀLEZ‐DUARTE Roser , JÖRNVALL Hans
Published: March 2005

Prokaryotic 3α/20β‐hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu‐C protease and trypsin (cleaving after Glu192 and Arg196, respectively). Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection. Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp‐N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu‐C and Asp‐N proteases. Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s).

Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation. With a 10‐fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251. Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification. Together, the proteolysis and chemical modifications highlight three segments of short‐chain dehydrogenase subunits, one mid‐chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C‐terminus.
Human placental alkaline phosphatase
Authors: CHANG Tsu‐Chung , HUANG Shih‐Ming , HUANG Ter‐Mei , CHANG Gu‐Gang
Published: March 2005

An improved method for the purification of human placental alkaline phosphatase is described. The partially purified enzyme from Sigma was further purified by successive Concanavalin A‐Sepharose and Q‐Sepharose chromatography. The whole procedure may be completed in one working day. Highly purified enzyme was obtained with a 39% yield. The intrinsic fluorescence of the enzyme decreased at elevated temperature. The conformation of the enzyme molecule was studied by the fluorescence quenching technique. Upward Stern‐Volmer plots were obtained for the quenching data which suggested that, in addition to collisional quenching, static quenching was involved in the quenching mechanism. The dynamic and static quenching constants were found to be 0.7 ± 0.16 M−1 and 0.44 ± 0.1 M−1, respectively, using acrylamide as the quenching agent. The corresponding values were 0.43 ± 0.23 M−1 and 0.84 ± 0.18 M−1, respectively, with KI as the quenching agent. Mg2+ and PO43− induced protein conformational changes which altered both the dynamic and static quenching constants. Mg2+ was found to be a non‐essential activator for the placental alkaline‐phosphatase‐catalyzed hydrolysis of 4‐nitrophenyl phosphate. At pH 9.8, Mg2+ increased Vmax by 1.2‐fold without affecting the Kd of the substrate. The tetranitromethane‐modified enzyme showed slower migration toward the anode on electrophoresis and increased Kd for Mg2+.
Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation
Authors: ALMAS Bjørg , LE BOURDELLES Beatrice , FLATMARK Torgeir , MALLET Jacques , HAAVIK Jan
Published: March 2005

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium‐binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP‐dependent protein kinase (Km= 5 μM, Vmax= 9.5 μmol · min−1· mg kinase−1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP‐dependent protein kinase by 2–3‐fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.