bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin‐dependent Na+ pump glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli
Authors: BENDRAT Klaus , BUCKEL Wolfgang
Published: March 2005

1. The primary sodium‐ion pump glutaconyl‐CoA decarboxylase (GCD) from Acidaminococcus fermentans is composed of four subunits: GCDA, the carboxytransferase (65 kDa), GCDB, the carboxylyase (36 kDa) GCDC, the biotin carrier (24 kDa) and GCDD (14 kDa) of unknown function. A genomic library of A. fermentans was screened with an antiserum raised against whole GCD. A clone giving the strongest reaction in an immunoassay contained a 12‐kbp genomic fragment from A. fermentans and was analysed further. An oligonucleotide deduced from the N‐terminus of GCDA was used for probing the corresponding gene gcdA. It is 1761 bp in length and encodes for a protein of 64.3 kDa. Both partial amino acid sequences obtained from GCDA, the N‐terminus as well as an internal tryptic peptide, were detected in the open reading frame (ORF) of gcdA.

2. Sequencing of the flanking regions revealed three adjacent ORF (ORF1–3) which do not code for any of the peptide sequences known of the other GCD subunits. The ORF downstream of gcdA (ORF3) is followed by hgdA and hgdB coding for 2‐hydroxyglutaryl‐CoA dehydratase, the preceeding enzyme of the pathway of glutamate fermentation. Our results suggest that at least these three genes of the hydroxyglutarate pathway are organised in an operon and that the genes of the other GCD subunits from which peptide sequences are known (GCDB and GCDC) are not located adjacent to gcdA.

3. gcdA was amplified from genomic DNA using the polymerase chain reaction and cloned into the expression vector pJF118HE. Active GCDA subunit (up to 2.8 nkat/mg protein), catalysing the biotin‐dependent formation of crotonyl‐CoA from glutaconyl‐CoA, was obtained in cell‐free extracts of Escherichia coli DH5α by moderately inducing the tac promoter of pJF118HE with 25–100 μM isopropyl‐1‐thio‐β‐d‐galactoside. Strong induction (1mM isopropyl‐1‐thio‐β‐d‐galactoside) led to the formation of inclusion bodies from which GCDA could not be reactivated. The apparent Km= 51 mM for free biotin of the expressed GCDA subunit with V= 1.9 nkat/mg protein is similar to that of butanol‐treated GCD composed of GCDA and GCDC (apparent Km= 40 mM). Biocytin was found to be a somewhat better carboxy acceptor for the expressed GCDA subunit (apparent Km= 13 mM; V= 1.0 nkat/mg protein).

4. Native GCD and expressed GCDA were treated with 2 mM N‐ethylmaleimide showing different kinetics of inactivation: GCD lost half of its activity within 6 min, whereas expressed GCDA required 21 min.
Biochemical, immunological and ultrastructural characterization of aggregation substances encoded by Enterococcus faeclis sex‐pheromone plasmids
Authors: HIRT Helmut , WANNER Gerhard , GALLI Dominique , WIRTH Reinhard
Published: March 2005

The sex‐pheromone system of Enterococcus faecalis can be viewed as a unique and highly efficient plasmid‐collection mechanism. The contact needed for transfer of the conjugative sexpheromone plasmids is mediated by an adhesin, called aggregation substance, which is encoded by these plasmids. We show here that for 17 of the 18 sex‐pheromone plasmids (pAM373 being the exception) described to date, their adhesins are immunologically related to each other. In each case, we observed the presence of an N‐terminal fragment of about 78 kDa in addition to the 137‐kDa form of mature aggregation substance. The cross‐reactions were different for the various plasmids. In the case of pPD1 the 78‐kDa fragment reacted only weakly. The aggregation substance encoded by sex‐pheromone plasmid pAD1 (Asa1) was characterized in detail. The conditions used for SDS/PAGE had a drastic influence on the migration behavior of mature aggregation substance and differently migrating, interconvertible forms were identified. Preliminary data indicate that Asa1 might be a glycoprotein. Antibodies were isolated which are directed against the N‐ and C‐terminal parts of aggregation substance. They showed about the same reactivity on Western biots; however, only antibodies directed against the N‐terminal part of the aggregation substance could inhibit the bacterial cell/cell contact. The reactions of the two antibody preparations with induced cells of E.faecalis was analyzed by transmission electron microscopy. The results indicated that especially the N‐terminal part of aggregation substance is exposed on the cell surface of E. faecalis; the C‐terminal part seems to be much less exposed.
Characterization of cardiac fatty‐acid‐binding protein from human placenta
Authors: DAS Tanya , SA Gaurisankar , MUKHERJEA Manju
Published: March 2005

When a 105 000Xg supernatant of human placenta was incubated with [1‐14C]oleate and subjected to Sephadex G‐75 gel filtration and HPLC, two fatty‐acid‐binding protein (FABP) peaks were obtained. One of these, when further purified by carboxymethyl‐cellulose, gave one 15.3‐kDa FABP with pI 5.3. The other, when chromatographed on DEAE cellulose, separated into two 14.2‐kDa FABP with pI 6.9 and 5.4. Purity of the proteins was checked by SDS/PAGE. Molecular mass, pI, immunochemical properties and amino acid compositions all indicated that 15.3‐kDa FABP was of the cardiac type, whereas both 14.2‐kDa FABP were of the hepatic type. Cardiac FABP did not cross‐react with hepatic proteins. When tested for the acceptor/donor properties of these FABP, hepatic types were found to be better candidates than cardiac in uptaking fatty acids from liposomes. Cardiac FABP, on the other hand acted in a more efficient way as a donor, indicating a distinct role of these proteins in human placenta, which furnishes a multiorgan system for the developing fetus.
Kinetics and mechanism of an NADPH‐dependent succinic semialdehyde reductase from bovine brain
Authors: CHO Sung‐Woo , SONG Min‐Sun , KIM Geum‐Yi , KANG Woo‐Don , CHOI Eui Yul , CHOI Soo Young
Published: March 2005

An NADPH‐dependent succinic semialdehyde reductase has been purified from bovine brain by several chromatographic procedures. The preparation appeared homogeneous on SDS/PAGE. The enzyme is a monomeric protein with a molecular mass of 28 kDa. A number of properties of the bovine brain enzyme, such as substrate specificity, specific activity, molecular mass, optimum pH, amino acid composition, and kinetic parameters, have been determined and compared with those reported for preparations from other sources. The results indicate that the enzyme isolated from bovine brain in the present study is different from those reported for preparations from other sources. The inhibition kinetic patterns obtained when the products of the reaction or substrate analogs are used as inhibitor of the reaction catalyzed by the enzyme are consistent with an ordered sequential mechanism involving the formation of an intermediate ternary complex and in which NADPH is the first substrate to bind the enzyme.
Differentially expressed patterns of glycosaminoglycan structure in heparan sulfate proteoglycans and free chains
Authors: HOVINGH Peter , PIEPKORN Michael , LINKER Alfred
Published: March 2005

The metabolic relationships between heparan sulfate proteoglycans, free chains, and oligosaccharides in different cell locations were evaluated by comparing their glycosaminoglycan structure. Metabolically labeled heparan sulfate proteoglycans of BALB/c 3T3 cell layers and in conditioned medium were compared with the heparan sulfate free chains (modal mass = 10 kDa) and oligosaccharides (modal mass = 3 kDa) of the cells. Nonlytic, in situ digestion with heparitinase I indicated that 90% of proteoglycans, 70% of the free chains, and 20% of the oligosaccharides were enzyme accessible, but there was no evidence using competitive iigands for binding of the products to the cell surface via the glycosaminoglycan moieties. Structurally, the membrane proteoglycans were the most O‐IN‐sulfated and yielded more tri‐ and tetra‐sulfated di‐ and tetra‐saccharides by nitrous acid degradation. In contrast, the side chanis of medium proteoglycans were less sulfated and more polydisperse in mass, suggesting that most medium proteoglycans are not processed from membrane precursors. The heparan sulfate free chains were of lower mass, less sulfated, and more heterogeneous in distribution of the anionic groups than were proteoglycan side chains. Corroborating analytical heparitinase I digestion indicted that generation of di‐ and tetra‐saccharides proportion‐ately increased from membrane proteoglycan, to cell free chain, to medium proteoglycan categories. Because the structural patterns of the heparan sulfate free chains did not reveal a clear relationship with the side chains of the major proteoglycans, their origin was further probed by [3H]BH4‐labeling of the reducing terminus under varying stringencies. The end‐labeled residues obtained by nitrous or strong acid hydrolysis of the free chains showed insignificant amounts of galactose and xylose, but rather glucosamine N‐sulfate and a residue likely generated from glucuronate. The effective labeling that was achieved with weak alkali indicated that covalent oligopeptide is not present. In summary, the heparan sulfate free chains, which in part are components of the cell surface, are of relatively low mass, are unassociated with covalent peptide, and most probably have a disaccharide motif of glucosamine N‐sulfate and a uronate residue at the reducing end. Taken together, these observations suggest that the free chains originate by processing of precursor heparan sulfate proteoglycans on the cell surface via an endoglycosidase acting on an N‐sulfated portoin of the original polymer.
Sequence‐specific 1H‐NMR assignment and secondary structure of black mamba dendrotoxin I, a highly selective blocker of voltage‐gated potassium channels
Authors: FORAY Marie‐Francoise , LANCELIN Jean‐Marc , HOLLECKER Michelle , MARION Dominique
Published: March 2005

The secondary structure of dendrotoxin I, an important constituent of the venom of the African black mamba snake Dendroaspis polylepis polylepis, was determined in aqueous solution by two‐dimensional methods. Complete sequence‐specific 1H‐NMR assignment was obtained with the exception of the backbone amide proton of Gly39 and Cys40. Dendrotoxin I is based on a central antiparallel β‐sheet and two small helices located at the N‐ and the C‐terminal extremities. These secondary‐structural units occur at exactly the same places in the amino acid sequence as those of bovine pancreatic trypsin inhibitor (BPTI), with which dendrotoxin I shares 33% sequence similarity. According to the disulfide‐bridge positions and the long‐range NOE observed these secondary‐structural elements fold in a similar manner to BPTI. This similarity allows an hypothesis according to which dendrotoxin I could derive from an ancestral Künitz‐type proteinase inhibitor. This ancestor would have been heavily mutated at amino acid positions not critical for gross structure. The spatial locations of the solvent‐exposed amino acids concerned could therefore serve as a guideline for interpretation of the structure/activity relationship of dendrotoxin I for the blockage of voltage‐sensitive potassium channels of which dendrotoxin I is a strong inhibitor. The possible connections with other polypeptide toxins that block related ion currents is discussed.
Cholesterol metabolism in rat adrenal gland during reversible endotoxic shock
Authors: ABARCA Santiago , GARCÍA Resurrección
Published: March 2005

The adrenal glands have a crucial role for survival during endotoxic shock. Cholesterol is the obligatory intermediary in corticosteroid biosynthesis; thus any alteration in either the availability of cholesterol or in the ability of the adrenal gland to use cholesterol would have a profound effect on corticosteriod production. We have studies the effect of Escherichia coli endotoxin on cholesterol metabolism, injecting lipopolysaccharide (1.6 mg/100g body) from E. coli 0111:B4 into the tail vein of male Wistar rat. Previous studies from this laboratory have shown that this dose of lipopolysaccharide induces a reversible endotoxic shock.

During reversible endotoxic shock there is an alteration in plasma cholesterol; plasma total‐cholesterol levels increase mainly at 6–24 h post‐lipopolysaccharide injection, whereas cholesterol in high‐density lipoproteins shows no significant variations, except a slight but significant decrease at 24 h. The cholesterol content in adrenal gland is diminished in endotoxemic rat, this decrease is more important at 6–24 h after endotoxin injection.

We have also measured the acyl‐CoA: cholesterol O‐acyltransferase (ACAT) and cholesterolesterase (CEH) activity during endotoxic shock. ACAT activity decreases after lipopolysaccharide injection. ACAT activity in endotoxemic rats is approximately 35–40% of the activity in control rats. This decrease is due to a defect in the functional capacity of the enzyme, since with exogenous cholesterol there is no significant variation in the ACAT activity. CEH activity, in contrast, increases during endotoxic shock: it shows a maximum (twofold the activity seen in control rats) at 6 h after lipopolysaccharide injection. These results show that lipopolysaccharide injection modifies cholesterol metabolism in plasma and in the adrenal gland, either directly or by mediators.
Cellular retinoic‐acid‐binding‐protein and retinol‐binding‐protein mRNA expression in the cells of the rat seminiferous tubules and their regulation by retinoids
Authors: FARAONIO Raffaella , GALDIERI Michela , COLANTUONI Vittorio
Published: March 2005

The levels of the mRNA corresponding to the intracellular binding proteins for retinoic acid and retinol (CRABP1 and CRBP1, respectively) were studied in primary cultures of somatic and germ cells of the rat seminiferous tubules. We show that the CRABP1 mRNA is expressed in Sertoli and germ cells and a single molecular species of mRNA is detected. CRBP1 mRNA is detected in Sertoli and peritubular cells. The regulation of the expression of both genes by retinoids was studied in Sertoli cells. CRABP1 mRNA levels are not affected by either retinoic acid or retinol, whereas both compounds positively regulate CRBP1 mRNA synthesis in a dose‐dependent manner. A fivefold increase in CRBP1 mRNA levels was observed 32–48 h after addition of either agent. These results demonstrate that in Sertoli cells the expression of CRABP1 is not affected by retinoids, similar to the situation observed in vivo and in other in‐vitro cultures. CRBP1‐gene expression is, instead, induced and the variations in CRBP1‐mRNA levels may regulate the intracellular concentrations of retinoids, as a response to changes in the vitamin‐A nutritional status.
The primary structure of turtle Cu,Zn superoxide dismutase
Authors: SCHININÀ M. Eugenia , BOSSA Francesco , LANIA Amalia , CAPO Concetta R. , CARLINI Patrizia , CALABRESE Lilia
Published: March 2005

A copper,zinc superoxide dismutase, has been isolated from the marine turtle Caretta caretta and the complete amino acid sequence obtained. The sequence was determined by isolation and analysis of peptides obtained after cleavage of the carboxymethylated apoenzyme with trypsin or Staphylococcus aureus protease. Turtle superoxide dismutase consists of 166 amino acid residues, which represents the largest number to date for a cytosolic copper,zinc superoxide dismutase. The comparison of this amino acid sequence with that of bovine superoxide dismutase revealed a oneresidue C‐terminal extension, two single residue insertions and a 12‐residue insertion in the N‐terminal region, in turtle superoxide dismutase. The new segment consists of a threefold repeating sequence and was found to be the site for selective proteolytic attack by trypsin under native conditions. The biochemical characteristics the spectroscopic and catalytic properties as well as the thermal stability and the resistance to irreversible denaturation, were carefully examined and were very similar to those of other superoxide dismutases. These results indicate that the presence of the new polypeptide segment does not affect the main folding of the chain and the quaternary structure, nor the functional parameters of turtle superoxide dismutase. The possibility that the new insert constitutes a loop excluded from the protein scaffold providing the framework of the active site is also discussed.
Affinity labelling of sorbitol dehydrogenase from sheep liver with α‐bromo‐β‐(5‐imidazolyl)propionic acid
Authors: REIERSEN Herald , SLETTEN Knut , McKINLEY‐McKEE John S.
Published: March 2005

The metal‐directed alkylating agent DL‐α‐bromo‐β‐(5‐imidazolyl)propionic acid (BrImPpOH) is shown to be an affinity‐labelling reagent for sheep liver sorbitol dehydrogenase (SDH). As previously found for horse liver alcohol dehydrogenase (ADH), it modifies a cysteine ligand to the active‐site zinc. In this case it is selectively incorporated (over 90%) at Cys43 in each of the four polypeptide chains/protomers of sheep liver SDH. Incorporated reagent and residual activity correlated. The first order inactivation constant, k2, and KEI, the dissociation constant for SDH and BrImPpOH, have been determined at different pH. The reactivity of BrImPpOH for SDH is higher than that for horse liver and yeast ADH. The protection of SDH against BrImPpOH inactivation by buffers and other molecules shows some similarities to that with horse liver ADH. However, sheep liver SDH bound BrImPpOH, imidazole and phosphate ions much weaker than liver ADH. The pKa values from the plot of log(k2/KEI) against pH are approximately 7.0 and 8.8–8.9. The former pKa value probably represents ionization of an imidazole group and the latter the zinc/water ionization in SDH. These pKa values are similar to those found for horse liver ADH. They are apparently not noticeably influenced by a second cysteine ligand in liver ADH being replaced by a proposed glutamic acid residue as a ligand to the catalytic zinc in SDH. The plot of logk2 against pH shows pKa values around 7.0 and 9.2 for the SDH‐BrImPpOH‐complex. The pKa of 7.0 is the same as for log(k2/KEI), and indicates no significant perturbation due to the binding of BrImPpOH to SDH. The pKa around 9.2 indicates perturbation of the zinc/water ionization or the ionization of Cys43.
Prechaperonin 60 and preornithine transcarbamylase share componunts of the import apparatus but have distinct maturation pathways in rat liver mitochondria
Authors: PERALTA Dadna , LITHGOW Trevor , HOOGENRAAD Nicholas J. , HØJ Peter B.
Published: March 2005

Mitochondrial preornithine transcarbamylase (p‐OTC) and premalate dehydrogenase (p‐MDH) are the only two matrix‐located preproteins so far identified for which the proteolytic processing in vitro requires the formation of genuine processinw intermediates, i‐OTC and i‐MDH, respectively. To establish the processing of other preproteins during import with respect to the two‐step processing of p‐PTC and p‐MDH, the chelators EDTA and 1,10‐phenanthroline were used to study the import and processing of rat prechaperonin 60 (p‐cpn60) and p‐OTC by mitochondria from four cpn60‐containing organs. We found no evidence for a secondary processing step in the maturation of p‐cpn60, but a clear requirement for two‐step processing of p‐OTC, even in three organs which do not contain ornithine transcarbamylase. The metal‐ion requirement of the p‐OTC processing activities in the organelle is consistent with the proposition that the mitochondrial processing protease (MPP) and mitochondrial intermediate peptidase (MIP) activities defined in vitro [Kalousek, F., Hendrick, J. P. & Rosenberg, L. E. (1988) Proc. Natl Acad. Sci. USA 85, 7536–7540] are responsible for precursor processing in vivo. The authenticity of two‐step processing in vivo was, furthermore, established by demonstrating that i‐OTC accumulates to high levels in Spodoptora frugiperda insect cells supplemented with MnCl2. The inability of the insest cells to process p‐OTC fully is not a characteristic of cells grown in culture since cultured rat hepatoma cells process p‐OTC fully processed m‐OTC. Finally, we find that the import and processing of p‐cpn60 and p‐OTC is inhibited in an identical fashion by presequence–bovine‐serum‐qlbumin conjugates. The differenses in proteolytic maturation between p‐cpn60 and p‐OTC are therefore not likely to result from different import pathways as the two precursors compete for common components of the import apparatus.
Dissociation of lactate dehydrogenase in aqueous and reversed micellar solutions
Authors: KHMELNITSKY Yuri L. , van HOEK Arie , VEEGER Cees , VISSER Antonie J. W. G.
Published: March 2005

Dissociation behavior of lactate dehydrogenase from hog muscle, both in aqueous solution and reversed micelles of sodium bis(2‐ethylhexyl sulfosuccinate) in octane was studied using time‐resolved polarized fluorescence spectroscopy. It was found that, in aqueous solutions, the enzyme underwent partial dissociation with the formation of isolated subunits at enzyme concentrations below 8 nM. Dissociation of the enzyme also took place upon entrapment of lactate dehydrogenase into reversed micelles under conditions of low surfactant hydration, when micelles were not large enough to accomodate a whole protein molecule.
Electrochemical studies of the hexaheme nitrite reductase from Desulfovibrio desulfuricans ATCC 27774
Authors: MORENO Cristina , COSTA Cristina , MOURA Isabel , LE GALL Jean , LIU Ming Y. , PAYNE William J. , van DIJK Cees , MOURA José J. G.
Published: March 2005

The electron‐transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfuricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second‐order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range 106–108 M−1 s−1 and increase in the direction D. desulfuricans cytochrome c3→D. vulgaris cytochrome c3→ methyl viologen. An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved.

Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility).

Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported.
Thymidine phosphorylase activity of platelet‐derived endothelial cell growth factor is responsible for endothelial cell mitogenicity
Authors: FINNIS Chris , DODSWORTH Neil , POLLITT Charles E. , CARR Grant , SLEEP Darrell
Published: March 2005

Recombinant human platelet‐derived endothelial cell growth factor, expressed in the yeast Saccharomyces cerevisiae was purified to greater than 98% purity by anion‐exchange and hydroxyapatite chromatography. It was shown to possess theymidine phosphorolytic activity in vitro (pH optiomum, pH 5.3; Km, 0.11 mM; Vmax, 12.5 mmol min−1 mg −1; turnover number, 9.4 s−1). Covalent modification simultaneously inhibited the enzymatic and mitogenic properties of the protein, while interaction with a cell‐surface receptor was not required to stimulate mitogenesis. Purified Escherichia coli thymidine phosphorylase was also mitogenic toward endothelial cells. It is proposed that platelet‐derived endothelial cell growth factor is human thymidine phosphorylase which promotes endothelial cell proliferation by reducing thymidine levels that would otherwise be inhibitory to endotheial cell growth.
β‐Actin mRNA‐binding proteins associated with the cytoskeletal framework
Authors: SHARPLESS Kathryn , BIEGEL Diane , YANG Tao , PACHTER Joel S.
Published: March 2005

Association of mRNA with the cytoskeletal framework (CSK) is thought to play a strategic role in the placement of mRNA in the cytoplasm. However, the molecular determinants underlying mRNA/CSK association are completely unknown. To begin addressing this issue, we have employed a binding assay to identify proteins of the CSK compartment of NIH 3T3 cells that bind in‐vitro‐transcribed 32P‐labelled β‐actin mRNA with high affinity. Three proteins, of approximate molecular masses 27, 50 and 97 kDa, were observed to exhibit strong binding. Binding to these proteins took place at physiological salt concentration and withstood washing in 0.5 M salt. Furthermore, binding was unaffected by heparin but was inhibited by unlabelled β‐actin mRNA. Treatment of isolated CSKs with the microfilament‐servering agent DNase I abolished all β‐actin mRNA‐binding activities, thus suggesting a possible association of β‐actin mRNA with the micorfilament network in situ. Removal of the 3′ untranslated region (UTR) significantly reduced β‐actin mRNA binding to all three CSK proteins but removal of the 5′ UTR mainly affected binding to the 97‐kDa species and that to a lesser extent. β‐Tubulin mRNA bound to the same three CSK proteins as did β‐actin mRNA, but with considerably less avidity. In contrast, vimentin mRNA strongly recognized these CSK proteins, and further bound to a group of smaller proteins (<29 kDa). As β‐actin mRNA, β‐tubulin mRNA and vimentin mRNA have been observed to occupy separate cytoplasmic locales, the proteins detected here may be operative both in binding mRNAs to the CSK in situ, as well as in localizing mRNA in the cytoplasm.
A step towards understanding the folding mechanism of horseradish peroxidase
Authors: PAPPA Helen S. , CASS Anthony E. G.
Published: March 2005

The guanidinium chloride denaturation/renaturation of the holo‐ and apo‐horseradish peroxidase isoenzyme c (HRP) has been studied by fluorescence and circular dichroism spectroscopies.

A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride (0.5 M). This intermediate has a secondary structure content like that of the native protein but a poorly defined tertiary structure. Renaturation of the apo‐HRP is reversible and 100% activity could be obtained after addition of a twofold excess of free haem.

The denaturation of the holo‐HRP is more complex and occurs in two distinct steps; unfolding of the protein backbone and loss of the haem. The denatured protein folds back to its native conformation but the incorporation of the haem occurs only after the secondary structure is formed.

Ca2+ appears to be important for the stability of the protein as the apo‐HRP is more resistant to denaturation in the presence of Ca2+. The free‐energy change during unfolding of the apo‐HRP was determined in the absence and presence of Ca2+ and found to be 9.2 kJ/mol and 16.7 kJ/mol, respectively. The importance of Ca2+ to the protein stability was also supported by studies on the loss of the haem from the protoporphyrin‐IX–apo‐HRP complex.
Nigerythrin and rubrerythrin from Desulfovibrio vulgaris each contain two mononuclear iron centers and two dinuclear iron clusters
Authors: PIERIK Antonio J. , WOLBERT Ronnie B. G. , PORTIER Gerrit L. , VERHAGEN Marc F. J. M. , HAGEN Wilfred R.
Published: March 2005

The trivial name ‘rubr‐erythrin’ is a contraction of two other trivial names: rubredoxin (ruber, red) and hemerythrin. It names a protein of undetermined biological function which putatively carries rubredoxin‐like mononuclear iron and hemerythrin‐like dinuclear iron. The name ‘nigerythrin’ (niger, black) is an analogy of rubrerythrin. It identifies a second protein of undetermined function which has prosthetic groups similar to rubrerythrin. Rubrerythrin was initially described [LeGall, J., Prickril, B. C., Moura, I., Xavier, A. V., Moura, J. J. G & Huynh, B.‐H. (1988) Biochemistry 27, 1636–1642] as a homodimer with four iron ions arranged into two rubredoxin sites and one inter‐subunit dinuclear cluster. Nigerythrin is a novel protein. Here, we report that both proteins are homodimers, each dimer carrying not four but six iron ions in two mononuclear centers and two dinuclear clusters. Rubrerythrin and nigerythrin are probably both located in the cytoplasm; they are differentially characterized with respect to molecular mass, pI, N‐terminal sequence, antibody cross‐reactivity, optical absorption, EPR spectrscopy, and reduction potentials. All three reduction potentials in both proteins are > +200 mV. These appear too high to be of practical relevance in the cytoplasm of the sulfate reducer Desulfovibrio vulgaris (Hildenborough). We suggest the possibility of a non‐redox role for both proteins with all six iron ions in the ferrous state.
Structural fluctuations between two conformational states of a transmembrane helical peptide are related to its channel‐forming properties in planar lipid membranes
Authors: VOGEL Horst , NILSSON Lennart , RIGLER Rudolf , MEDER Sabine , BOHEIM Günther , BECK Werner , KURTH Hans‐Hagen , JUNG Günther
Published: March 2005

Putative transmembrane helices of membrane proteins in general and channel proteins in particular often contain proline residues which may induce a bend into an otherwise regular helical structure. Here we show by fluorescence‐energy‐transfer measurements and molecular‐dynamics calculations that, in the case of synthetic bilayer‐spanning helical polypeptides, a proline‐induced bend in a helix acts as a flexible element mediating rigid body motions of the helical segments. Most important, such structural fluctuations in the transmembrane helices seem to play a functional role in the formation of ionic channels in planar lipid bilayers and biological membranes.
Cloning, sequencing, and expression of Trypanosoma brucei dihydrolipoamide dehydrogenase
Authors: ELSE Anthony J. , HOUGH David W. , DANSON Michael J.
Published: March 2005

A gene encoding dihydrolipoamide dehydrogenase was isolated from Trypanosoma brucei genomic DNA by using a combination of polymerase chain reaction and screening of a λ EMBL3 library. The DNA sequence reveals that it encodes a protein of 478 amino acids (Mr 49935) highly similar to previously sequenced dihydrolipoamide dehydrogenases. The gene was ligated into pMEX8 and expressed in an Escherichia coli mutant that lacks dihydrolipoamide dehydrogenase. Expression resulted in the appearance of dihydrolipoamide dehydrogenase activity concurrent with the production of a protein of the expected Mr as determined by SDS/PAGE and Western blotting.
Purification and characterization of anthranilate synthase from Catharanthus roseus
Authors: POULSEN Charlotte , BONGAERTS Roger J. M. , VERPOORTE Robert
Published: March 2005

Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of Catharanthus roseus by poly(ethylene glycol) precipitation/fractionation and subsequent separation by anion exchange on Q‐Sepharose, Orange A dye chromatography, Mono Q anion‐exchange chromatography and Superose 6 gel filtration. By analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5±0.5 kDa, respectively. The molecular mass determined by gel filtration was 143±5 kDa. The enzyme had a pI of 5.1 determined by chromatofocusing. The pH optimum was between pH 7.5 and pH 8.3, but the type of buffer used affected the results. The enzyme could utilize NH4+ as ammonium donor instead of glutamine. The enzyme showed normal Michaelis‐Menten kinetics with respect to the substrates l‐glutamine and chorismate, and the cofactor Mg2+, Km values for l‐glutamine was determined to be 0.37±0.05 mM, for chorismate 67±3 μM, and for MgCl2 0.26±0.03 mM respectively. Anthranilate synthase was inhibited by l‐tryptophan, tryptamine and d‐tryptophan (with l‐tryptophan being the best inhibitor). The enzyme was allosterically regulated showing positive cooperativity of chorismate binding at higher concentrations of tryptophan. For a tryptophan concentration of 20 μM the Hill coefficient was determined to be 2. The tryptophan binding sites showed positive cooperativity for higher concentrations of chorismate. The purified enzyme did not contain anthranilate‐5‐phosphoribosylpyrophosphate phosphoribosyltransferase activity and is thus not of the same type as the well characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl pyrophosphate transferase bifunctional type.
Epidermal‐growth‐factor receptors generate Ras · GTP more efficiently than insulin receptors
Authors: OSTEROP Arthur P. R. M. , MEDEMA René H. , v. d. ZON Gerard C. M. , BOS Johannes L. , MÖLLER Wim , MAASSEN J. Antonie
Published: March 2005

Activation of the Ras proto‐oncogene contributes in general to mitogenic activation of cells.

We show here that epidermal growth factor (EGF) stimulates Ras · GTP formation very efficiently in a variety of cell lines expressing endogenous EGF receptors only. Maximal activation of the receptor converts up to 65% of cellular p21ras from the GDP form into the active GTP‐bound state. This efficient activation occurs also in cultured primary human fibroblasts. Maximal insulin‐induced Ras · GTP formation is less but in cells overexpressing the insulin receptor a similar high response of Ras · GTP formation is observed after insulin stimulation.

Not only the efficiency but also the kinetics by which the EGF and insulin receptors stimulate Ras · GTP formation are quite distinct. In the Rat‐1‐derived cell line, H13IR2000, overexpressing both p21Ha‐ras and the insulin receptor, the activated insulin receptor generates approximately 1 mol Ras · GTP/mol activated insulin receptor. The activated EGF receptor amplifies the signal, resulting in the activation of approximately 40 mol p21ras/mol receptor.

Moreover, EGF‐stimulated generation of Ras · GTP is transient with a maximum after 2 min of hormone stimulation and diminishes to near basal levels within 1 h whereas the insulin‐induced Ras · GTP levels are maximal at 5–10 min and decline only slowly to half‐maximal in 1 h.

Desensitization of the EGF pathway by prolonged EGF stimulation, prevents subsequent stimulation of Ras · GTP formation by newly added EGF but not by insulin. Vice versa, in cells preincubated with insulin for 1 h, EGF stimulates Ras · GTP formation to near maximal values. These observations indicate that desensitization by prolonged hormone incubation does not involve inactivation of common signaling intermediates but rather components, specific for each pathway, like the particular receptors. The rapid down regulation of EGF receptors compared to insulin receptors corroborate this possibility.

The observed high potency of EGF receptors to generate Ras · GTP may explain the, in general stronger mitogenic activity of EGF compared to insulin.
Study of the states and populations of the rat pancreatic cholecystokinin receptor using the full peptide antagonist JMV 179
Authors: SILVENTE POIROT Sandrine , HADJIIVANOVA Christina , ESCRIEUT Chantal , DUFRESNE Marlène , MARTINEZ Jean , VAYSSE Nicole , FOURMY Daniel
Published: March 2005

The full peptide antagonist of the pancreatic cholecystokinin (CCK) receptor, JMV 179, [Boc‐Tyr(SO3H)‐Ahx‐Gly‐dTrp‐Ahx‐Asp phenylethyl ester, where Tyr(SO3H) = sulfated tyrosine, Ahx = 6‐aminohexanoic acid] was modified at its N‐terminus by incorporation of p‐hydroxyphenyl propionate (Bolton‐Hunter reagent, BH) and was subsequently radioiodinated. After HPLC purification, 125I‐BH‐JMV‐179, a CCK antagonist radioligand of high specific activity (2000 Ci/mmol) was obtained. 125I‐BH‐JMV‐179 bound to a single population of sites on rat pancreatic plasma membranes, (Kd= 3.9 nM, Bmax= 40 pmol/mg protein). Binding was dependent on time, temperature, and protein concentration, and was fully reversible. JMV 179 radioligand detected four times as many sites as an agonist radioligand [C. Hadjiivanova, M. Dufresne, S. Poirot, P. Sozzani, N. Vaysse, L. Moroder and D. Fourmy (1992) Eur. J. Biochem. 204, 273–279]. Agonists and antagonists of the A‐ and B‐subtype CCK/gastrin receptors inhibited 125I‐BH‐JMV‐179 binding with an order of potency compatible with the A‐subtype CCK receptor pharmacology. Moreover, the sulfate group on the tyrosine residue of the CCK peptides appeared to be of much less importance for antagonist affinity than for agonist affinity. Inhibition of 125I‐BH‐JMV‐179 binding by agonists (except JMV 180), demonstrated the presence of two affinity classes of binding sites. The population of sites having an apparent high affinity for CCK represented 30 pmol/mg protein and threefold the number of high‐affinity sites previously identified by an agonist radioligand. In presence of non‐hydrolyzable GTP, all the sites bound CCK agonists with a low affinity. Moreover, saturation analysis of JMV 179 radioligand binding in the presence of CCK indicated that CCK interacted competitively with all JMV 179 sites and demonstrated binding of JMV 179 radioligand to two distinct affinity classes of sites. In the presence of GTP[S] a single affinity class of sites for JMV 179 radioligand was found as in the control experiments without CCK.

This study, with the first CCK peptide antagonist radioligand, demonstrtes that CCK receptors exist in two interconvertible affinity states regulated by guanine‐nucleotide‐binding regulatory protein(s) in rat pancreatic plasma membranes. JMV 179 radioligand does not induce receptor coupling but distinguishes the two affinity states of the CCK receptors. JMV 179 reveals the existence of populations of high‐affinity and low‐affinity site for CCK which had not previously been detected by agonist radioligand binding, thus suggesting heterogeneity of CCK receptor sites in membranes.
Purification and characterization of the major 50‐kDa repressor protein from cytoplasmic mRNP of rabbit reticulocytes
Authors: MINICH Waldemar B. , MAIDEBURA Igor P. , OVCHINNIKOV Lev P.
Published: March 2005

A 50‐kDa protein has been purified to homogeneity from free mRNP of rabbit reticulocytes. This protein, designated as p50, is present within both free mRNP (approximately 4 mol protein/mol globin mRNA) and polyribosomal mRNP (approximately 2 mol protein/mol globin mRNA). p50 is a basic protein (pI ∼ 9.5) and is characterized by a high glycine content of approximately 20%. Nitrocellulose‐filter analysis has shown that p50 interacts with globin mRNA with an association constant of approximately 2.5 × 108 M−1 (100 mM KAc, 4°C). Various RNA and polyribonucleotides have the following relative affinity for p50; poly(G) > poly(U) > globin mRNA ∼ 16S rRNA > poly(A) > poly(C). p50 can be phosphorylated both in vitro and in vivo.
Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K2tu‐PA expressed in Chinese hamster ovary cells
Authors: BERGWERFF Aldert A. , van OOSTRUM Jan , ASSELBERGS Fred A. M. , BÜRGI Rolf , HOKKE Cornelis H. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G.
Published: March 2005

A recombinant human plasminogen activator hybrid variant K2tu‐PA, expressed in Chinese hamster ovary cells, is partially glycosylated at Asn12 (A chain, kringle‐2 domain) and completely glycosylated at Asn247 (B chain, protease domain). After release of the N‐linked carbohydrate chains by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the protein by gel permeation chromatography, then fractionated by FPLC on Mono Q, followed by HPLC on Lichrosorb‐NH2, and analysed by 500‐MHz 1H‐NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (8%), disialylated diantennary (45%), disialylated tri‐ and trí‐antennary (1%), trisialylated tri‐ and trí‐antennary (28%), and tetrasialylated tetra‐antennary (18%) structures, all having fucose in α(1‐6)‐linkage at the Asn‐bound N‐acetylglucosamine. Sialic acid occurred exclusively in α(2‐3)‐linkage to galactose, and consisted of N‐acetylneuraminic acid (94%), N‐glycolylneuraminic acid (3%), and N‐acetyl‐9‐O‐acetylneuram‐inic acid (3%). In addition, glycopeptide fragments corresponding with the A or B chain of K2tu‐PA were analysed. The oligosaccharides attached to Asn12 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K2tu‐PA with that of tissue‐type plasminogen activator from different biological sources showed significant differences.

Profiling studies on different K2tu‐PA production batches demonstrated that the structures of N‐linked oligosaccharides were identical, but that relative amounts vary with the applied isolation procedure of the chimeric glycoprotein.
Interaction of ATP analogs with yeast 3‐phosphoglycerate kinase
Authors: PINEDA Teresa , KWON Oh‐Shin , SERPERSU Engin H. , CHURCHICH Jorge E.
Published: March 2005

Yeast 3‐phosphoglycerate kinase is inactivated by incubation with pyridoxal 5′‐diphospho‐5′‐adenosine (AdoP2Pxy) [Tamura, J. K., Rakov, R. D. & Gross, R. L. (1986) J. Biol. Chem. 261, 4126–4133). Incorporation of 1 mol affinity label/mol enzyme was sufficient for complete inactivation of 3‐phosphoglycerate kinase. The substrate ATP affords substantial protection against inactivation. Partial protection is afforded by the substrate glycerate 3‐phosphate.

When AdoP2Pxy‐modified phosphoglycerate kinase was reduced with [3H]NaBH4 and subjected to trypsin hydrolysis, only one radioactive peptide was isolated by reverse‐phase high‐performance liquid chromatography. The amino acid composition and sequence analysis of the purified radioactive peptide revealed that it spans residues 379–403 of the enzyme and Lys385 specifically reacted with the affinity label. This peptide represents the hinge region between the two domains of the protein, where the active site is also located.

The fluorescence intensity of enzyme‐bound AdoP2Pxy is enhanced when glycerate 3‐phosphate is added, suggesting exposure of the fluorescent probe to a more hydrophobic environment. Another fluorescent analog, anthraniloyl‐dATP (ant‐dATP), which carries the fluorescent reporter group on the ribose ring, binds to the enzyme at two distinct sites with Kd values of 6±2 μM and 25±3 μM, as determined by steady‐state anisotropy measurements. Bound ant‐dATP was displaced from the enzyme by glycerate 3‐phosphate and ATP, as monitored by the fluorescence anisotropy.

These results suggest that both fluorescent ATP analogs bind to the active site, which is at the hinge region of the enzyme. Model‐building studies showed that when AdoP2Pxy is built into the open form of the enzyme, as described in X‐ray studies, the pyridoxyl group of AdoP2Pxy cannot reach Lys385 for Schiff‐base formation. Labeled Lys385 is on a β‐turn immediately following helix XII, which was suggested to interact with the nucleotide and become ordered at the active site of 3‐phosphoglycerate kinase [Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kinesman, A. J. & Kinesman, S. M. (1982) EMBO J. l, 1635–1640]. The results presented here suggest that binding of substrates cause significant structural changes in the enzyme.
Molecular cloning and expression of the gene for serine hydroxymethyltransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2
Authors: MIYATA Atsuro , YOSHIDA Toyokazu , YAMAGUCHI Kenji , YOKOYAMA Chieko , TANABE Tadashi , TOH Hiroyuki , MITSUNAGA Toshio , IZUMI Yoshikazu
Published: March 2005

The gene encoding serine hydroxymethyltransferase (SHMT), one of the key enzymes of the one‐carbon‐compound assimilation of a methylotroph, Hyphomicrobium methylovorum GM2, and its flanking regions were isolated using a DNA fragment encoding Escherichia coli SHMT as a probe. Nucleotide sequencing of the recombinant plasmids revealed the SHMT gene codes for the 434‐amino‐acid protein with a calculated molecular mass of 46 068 Da. The amino‐acid sequence of the enzyme showed identity to the sequences of the enzymes from E. coli (55%) and rabbit liver (44%). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223‐3, was introduced to an SHMT‐deficient E. coli mutant ME5427 (glyA−). The transformed E. coli cells expressed SHMT, which was immunologically and enzymologically indistinguishable from the enzyme isolated from H. methylovorum GM2.
Ascidian entactin/nidogen
Authors: NAKAE Hiroki , SUGANO Mitsuko , ISHIMORI Yoshio , ENDO Takeshi , OBINATA Takashi
Published: March 2005

Entactin/nidogen, a major component of the basement membrane, has a domain structure comprising three globular domains, and thread‐like and rod‐like domains connecting them. It contains six epidermal‐growth‐factor‐(EGF)‐like motifs and one thyroglobulin‐like motif.

In the present study, ascidian entactin/nidogen has been identified by a monoclonal antibody technique. We prepared anti‐(ascidian entactin/nidogen)IgG, named anti‐AsEnt1, then cloned the cDNA of ascidian entactin/nidogen using anti‐AsEnt1 as a probe, and determined its entire sequence. Mainly because the deduced amino acid sequence exhibited high similarity to mouse entactin and human nidogen, and because the antigen localized in basement membrane of ascidian body‐wall muscle, we have concluded that the antigen anti‐AsEnt1 corresponds to the ascidian entactin/nidogen homologue.

The deduced amino acid sequence of ascidian entactin/nidogen clearly showed that the ascidian homologue also has a domain structure. However, the ascidian homologue lacked the thread‐like domain, and the rod‐like domain differed from that of mouse entactin in composition, consisting of two kinds of cysteine‐rich motifs, that is, the EGF‐like motif and the thyroglobulin‐like motif. These results suggest that entactin/nidogen have evolved by modifying the domains, especially by shuffling the two kinds of cysteine‐rich motifs.
Functional expression and characterization of a Xenopus laevis peptidylglycine α‐amidating monooxygenase, AE‐II, in insect‐cell culture
Authors: SUZUKI Kenji , OHTA Mitsue , OKAMOTO Megumi , NISHIKAWA Yoshiki
Published: March 2005

The α‐amidating reaction of peptide hormones is a two‐step process which is catalyzed by peptidylglycine α‐hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N‐C lyase (PHL). There are three types of mRNA for these amidating enzymes in Xenopus laevis, namely AE‐I, AE‐II and AE‐III. AE‐I encodes only PHM and AE‐III encodes both PHM and PHL. AE‐II seems to encode subtypes of both PHM and PHL. While AE‐II mRNA is present in high amounts in frog skin, the actual enzymes originating from AE‐II have not been detected. When we expressed AE‐II in cultured insect‐cells using the baculovirus expression vector system, the expressed enzyme was specifically localized to the membrane fraction due to its hydrophobic transmembrane domain. Alternatively, when the transmembrane‐domain‐deleted AE‐II (Met1–Ile731) was expressed, the enzyme was secreted into the culture medium; this secreted enzyme was purified to homogeneity by a simple two‐step procedure. We have verified that the reaction product of the purified enzyme was the amidated peptide, indicating that AE‐II has the ability to catalyze the entire amidating reaction.