bluuebunny/crossref_metadata_2025 huggingface dataset:
The pur8 gene from the pur cluster of Streptomyces alboniger encodes a highly hydrophobic polypeptide which confers resistance to puromycin
Authors: TERCERO José Antonio , LACALLE Rosa Ana , JIMENEZ Antonio
Published: March 2005

A novel puromycin‐resistancer determinant (pur8) was isolated from one end of the pur cluster that encodes the puromycin biosynthetic pathway from Streptomyces alboniger and expressed in Streptomyces lividans. The gene pur8 induced antibiotic resistance that was highly specific for puromycin. The nucleotide sequence of pur8 contains an open reading frame of 1512 bp whose deduced amino acid sequence encodes a polypeptide (Pur8) with 14 possible transmembrane spanning segments. It shows significant similarities to other known or putative transmembrane proteins, including a number which confer drug resistance in a variety of antibiotic‐producing Streptomyces, Gram‐positive and Gram‐negative bacteria, and some solute transporters of prokaryotic and eukaryotic origin. As is probably the case for most of these proteins, Pur8 may be involved in active puromycin efflux energized by a proton‐dependent electrochemical gradient. In addition, it could be implicated in secreting N‐acetylpuromycin, the last intermediate of the biosynthesis pathway, to the environment.
Flavin dynamics in oxidized Clostridium beijerinckii flavodoxin as assessed by time‐resolved polarized fluorescence
Authors: LEENDERS Rik , VAN HOEK Arie , VAN IERSEL Martijn , VEEGER Cees , VISSER Antonie J. W. G.
Published: March 2005

The time‐resolved fluorescence characteristics of flavin in oxidized flavodoxin isolated from the anaerobic bacterium Clostridium beijerinckii have been examined. The fluorescence intensity decays were analyzed using the maximum‐entropy method. It is demonstrated that there exist large differences in fluorescence behaviour between free and protein‐bound FMN. Three fluorescence lifetime components are found in oxidized flavodoxin, two of which are not present in the fluorescence‐intensity decay of free FMN. The main component is distributed at 30 ps, with relative contribution of 90%. Another minor component (4% contribution) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluoresence decay of free FMN. The results allowed us to determine the dissociation constant, Kd= 2.61×10−10 M (at 20°C).

Conllisional fluorescence‐quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partially, exposed to the solvent. The shortest lifetime is not affected significantly, indicating that it possibly originates from an active‐site conformation in which the flavin is more or less buried in the protein and not accessible to iodide.

The fluorescence anisotropy behaviour of free and protein‐bound FMN was examined and analyzed with the maximum‐entropy method. It was found that an excess of apoflavodoxin is required to detect differences between free and protein‐bound FMN. In free FMN one single distribution of rotational correlation times is detected, whereas in flavodoxin the anisotropy decay is composed of more than one distribution.

Associative analysis of fluorescence anisotropy decays shows that part of the 4.8 ns fluorescence lifetime present in the flavodoxin fluorescence decay, is coupled to a rotational correlation time similar to that of free FMN in solution, while another part of this lifetime is coupled to a longer correlation time of about 1 ns. This finding is in accordance with earlier studies [Barman, B. G. & Tollin, G. (1972) Biochemistry 11, 4746–4754] in which it was proposed that the first binding step of the flavin to the protein involves the phosphate group rather than another part of the FMN.

The two shortest fluorescence lifetimes, which do not carry information on the long‐term rotational behaviour of the protein, seem nonetheless to be associated with a longer rotational correlation time which is comparable to overall protein tumbling. These lifetime components probably originate from a complex in which the flavin‐ring system is more or less immobilized within the protein matrix.
Conformational differences between complexes of elongation factor Tu studied by 19F‐NMR spectroscopy
Authors: ECCLESTON John F. , MOLLOY David P. , HINDS Mark G. , KING Rodney W. , FEENEY James
Published: March 2005

An analogue of elongation factor Tu (EF‐Tu) from Escherichia coli was prepared by biosynthetic incorporation of 3‐fluorotyrosine. The 19F‐NMR spectra of the binary complexes of this protein with GDP, GTP and elongation factor Ts (EF‐Ts) and the ternary complexes EF‐Tu · GDP · aurodox and EF‐Tu · GDP · EF‐Ts were measured. EF‐Tu contains ten tyrosine residues and all of the complexes studied gave complex 19F spectra with overlapping resonances. EF‐Tu · GDP gave a spectrum in which two signals were markedly different from those shown by the other complexes, the two resonances being shifted downfield by at least 3.4 ppm and 0.9 ppm relative to their shifts in the other complexes. Such large downfield shifts can be explained by second‐order electric field shielding effects resulting from these two tyrosine residues being in a sterically constrained environment in EF‐Tu · GDP and with the steric restraints being released in all of the other complexes. The X‐ray diffraction structure of EF‐Tu · GDP shows that Tyr87 in the N‐terminal domain (domain I) and Tyr309 in the C‐terminal domain (domain III) are both buried within the protein and are close to each other: these residues are in regions of EF‐Tu previously implicated in the structural changes between EF‐Tu · GDP and EF‐Tu · GTP by other workers. If these tyrosine residues correspond to the two downfield resonances of the spectra of EF‐Tu · GDP, the results from the 19F‐NMR would be consistent with these earlier indications that domain I interacts closely with domain III in EF‐Tu · GDP and that the amino acids between Gly83 and Gly100 are an important part of this interaction. For all the other complexes studied, these tyrosines are in a less sterically crowded environment consistent with a weaker interaction between the two domains. The 19F‐NMR spectrum of the trypsin‐cleaved product of EF‐Tu · GDP, from which the X‐ray diffraction structural data have been obtained, shows no significant differences from the native protein so that trypsin cleavage causes no large changes in the protein's structure.
Unfolding/refolding studies of the myosin rod
Authors: NOZAIS Muriel , BECHET Jean‐Jacques
Published: March 2005

The effect of guanidine hydrochloride on the gel‐filtration chromatography, viscosity, far ultraviolet circular dichroism and fluorescence emission intensity of the myosin rod was studied under equilibrium conditions. The normalized transition curves for each of these methods were comparable with a midpoint at a guanidine hydrochloride concentration of 1.75–2 M. The curves were not, however, superposable, suggesting that the loss of helix content and the dissociation of the two chains of the myosin rod were not tightly linked. Furthermore, they were unexpectedly independent of the protein concentration over 0.05–20 μM. These phenomena are interpreted taking into account the large size of the molecule. A step‐wise process is proposed as a model for the unfolding of the myosin rod.
Effect of side‐chain structure on inhibition of yeast fatty‐acid synthase by cerulenin analogues
Authors: MORISAKI Naoko , FUNABASHI Hiroshi , SHIMAZAWA Rumiko , FURUKAWA Jun , KAWAGUCHI Akihiko , OKUDA Shigenobu , IWASAKI Shigeo
Published: March 2005

Yeast fatty‐acid synthase (FAS) inhibition by cerulenin analogs with varying side‐chain lengths was compared with that of cerulenin, tetrahydrocerulenin and iodoacetamide. Although inhibition by cerulenin was the highest, the analogs having (E,E)‐Δ7,10 double bonds showed high inhibition. This strongly suggests that the (E, E)‐λ7.10 double bonds play an important role in the interaction of the inhibitors with the enzyme. It was suggested that the size of the hydrophobic cavity in the condensing enzyme terminates fatty‐acid chain elongation by decreasing inhibition by the C18 analog. Like cerulenin itself, the shortest analog (C6) did not induce malonyl‐CoA decarboxylase activity.
Doxorubicin‐induced lipid peroxidation and glutathione peroxidase activity in tumor cell lines selected for resistance to doxorubicin
Authors: BENCHEKROUN M. Nabil , POURQUIER Philippe , SCHOTT Brigitte , ROBERT Jacques
Published: March 2005

Doxorubicin‐induced lipid peroxidation was evaluated in four human or murine cell strains in culture and in their doxorubicin‐resistant variants, by the quantification of malondialdehyde produced after a 2‐h incubation of cells with the drug. Significantly increased malondialdehyde levels were obtained 24 h after doxorubicin treatment in three of the wild‐type cell lines with doses as low as 0.05–0.1 μg/ml, which is within an order of magnitude of the concentration of the drug which inhibits cell growth by 50%. This production of malondialdehyde was abolished in two doxorubicinresistant strains, even with high doses of drug (100–300 μg/ml), but was maintained in the third resistant line. No malondialdehyde production was observed in the fourth cell line, sensitive or resistant. It is remarkable that an enhancement of selenium‐dependent and non‐selenium‐dependent glutathione peroxidase activities was exhibited during the acquisition of resistance to doxorubicin in the two first lines, but not in the third, whereas a constitutively high non‐selenium‐dependent glutathione peroxidase activity existed in the doxorubicin‐sensitive and doxorubicin‐resistant variants of the fourth cell line. Gene expression of selenium‐dependent glutathione peroxidase and of glutathione S‐transferase π, which is known partially to bear a non‐selenium‐dependent glutathione peroxidase activity, were correlated with the corresponding enzyme activities. It appears, therefore, that the already known enhancement of glutathione peroxidase activity and expression in doxorubicin‐resistant cell lines has a quantifiable consequence upon doxorubicin‐induced lipid peroxidation and may have consequences in the mechanism of resistance to this drug.
Characterization of prostaglandin‐F 2α‐binding sites on rat hepatocyte plasma membranes
Authors: NEUSCHÄFER‐RUBE Frank , PÜSCHEL Gerhard P. , JUNGERMANN Kurt
Published: March 2005

Prostaglandin (PG) F 2α has previously been shown to increase glucose output from perfused livers and isolated hepatocytes, where it stimulated glycogen phosphorylase via an inositol‐trisphosphate‐dependent signal pathway. In this study, PGF 2α binding sites on hepatocyte plasma membranes, that might represent the putative receptor, were characterized.

Binding studies could not be performed with intact hepatocytes, because PGF 2α accumulated within the cells even at 47deg;C. The intracellular accumulation was an order of magnitude higher than binding to plasma membranes.

Purified hepatocyte plasma membranes had a high‐affinity/low‐capacity and a low‐affinity/high‐capacity binding site for PGF 2α. The respective binding constants for the high‐affinity site were Kd= 3 nM and Bmax= 6 fmol/mg membrane protein, and for the low‐affinity site Kd= 426 nM and Bmax= 245 fmol/mg membrane protein.

Specific PGF2α binding to the low‐affinity site, but not to the high‐affinity site, could be enhanced most potently by GTP[γS] followed by GDP[βS] and GTP, but not by ATP[γS] or GMP.

PGF2α competed most potently with [3H]PGF 2α for specific binding to hepatocyte plasma membrances, followed by PGD2 and PGE2.

Since the low‐affinity PGF2α‐binding site had a Kd in the concentration range in which PG had previously been shown to be half‐maximally active, and since this binding site showed a sensitivity to GTP, it is concluded that it might represent the receptor involved in the PGF2α signal chain in hepatocytes. A biological function of the high‐affinity site is currently not known.
Specificity of T‐cell protein tyrosine phosphatase toward phosphorylated synthetic peptides
Authors: RUZZENE Maria , DONELLA‐DEANA Arianna , MARIN Oriano , PERICH John W. , RUZZA Paolo , BORIN Gianfranco , CALDERAN Andrea , PINNA Lorenzo A.
Published: March 2005

The local specificity determinants for a T‐cell protein tyrosine phosphatase (TC‐PTP) have been inspected with the aid of a series of synthetic peptides, either enzymically or chemically phosphorylated, derived from the phosphoacceptor sites of phosphotyrosyl proteins. The truncated form of T‐cell PTP, deprived of its C‐terminal down‐regulatory domain, readily dephosphorylates submicromolar concentrations of eptapeptides to eicosapeptides, reproducing the C‐terminal down‐regulatory site of pp60c‐src (Tyr527), the phosphorylated loop IV of calmodulin and the main autophosphorylation site of two protein tyrosine kinases of the src family (Tyr416 of pp60c‐src and Tyr412 of p51fgr). However, phosphopeptides of similar size, derived from phosphoacceptor tyrosyl sites of the abl and epidermal‐growth‐factor receptor protein tyrosine kinases, the phosphorylated loop III of calmodulin, and phosphoangiotensin II undergo either very slow or undetectable dephosphorylation, even if tested up to 1 μM concentration.

The replacement of either Ser‐P or O‐methylated phosphotyrosine for phosphotyrosine within suitable peptide substrates gives rise to totally inert derivatives. Moreover, amino acid substitutions around phosphotyrosine in the peptides src‐(412–418), src‐(414–418) and abl‐(390–397) deeply influence the dephosphorylation efficiency. From these data and from a comparative analysis of efficient versus poor phosphopeptide substrates, it can be concluded that acidic residues located on the N‐terminal side of phosphotyrosine, with special reference to position –3, play a crucial role in substrate recognition, while basic residues in the same positions act as negative determinants. In any event, the presence of at least two aminoacyl residues upstream of phosphotyrosine represents a necessary, albeit not sufficient, condition for detectable dephosphorylation to occur.

By replacing the truncated form of TC‐PTP with the full length TC‐PTP, the dephosphorylation efficiencies of all peptides tested are dramatically impaired. Such an effect is invariably accounted for by a substantial increase in Km values, accompanied by a more or less pronounced decrease in Vmax values. These data support the concept that the C‐terminal regulatory domain of TC‐PTP exerts its function primarily by altering the affinity of the enzyme toward its phosphotyrosyl targets.
Characterization and kinetic properties of a soya‐bean cell‐wall phosphatase
Authors: FERTÉ Nathalie , MOUSTACAS Anne‐Marie , NARI Joannès , TEISSERE Marcel , BOREL Maguy , THIEBART Isabelle , NOAT Georges
Published: March 2005

A phosphatase from soya‐bean cell walls was purified to homogeneity and characterized. It consists of two identical 70‐kDa subunits linked by one or several disulphide bridges and, to our knowledge, it does not seem to require metal ions to be fully active. At high substrate concentrations, the enzyme was most efficient at slightly alkaline pH levels, which is at variance with the acid requirements of phosphatases previously established in other plant cell walls; whereas at low substrate concentrations it was more active at acid pH levels. The results of kinetic studies suggested that three ionizable groups of the protein might take part in the reaction. This enzyme is able to hydrolyse various phosphate esters, including PCho and nucleotides. Comparisons between the properties of the enzyme bound to the cell walls to those of the purified enzyme suggest that it is the most common, and perhaps the sole, phosphatase present in soya‐bean cell walls.
Ribonucleases from the extreme thermophilic archaebacterium S. solfataricus
Authors: FUSI Paola , TEDESCHI Gabriella , ALIVERTI Alessandro , RONCHI Severino , TORTORA Paolo , GUERRITORE Andrea
Published: March 2005

A purification procedure consisting of DEAE‐Sephacel chromatography, heparin‐Sepharose CL‐6B chromatography and Mono‐S chromatography led to the isolation of three proteins endowed with RNase activity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. They were referred to as p1, p2 and p3, according to their elution order from the Mono‐S column. Complete amino acid sequence of p2 and partial sequence of p3 displayed high sequence similarity to the 7‐kDa DNA‐binding proteins previously isolated in Sulfolobus strains [Choli, T., Wittman‐Liebold, B. & Reinhardt, R. (1988) J. Biol. Chem. 263, 7087–7093]. The molecular mass of p2, calculated from sequence data, was 7.02 kDa, which compares fairly well with the value of 7.4 kDa determined by SDS/PAGE. Gel filtration of the molecule under native conditions displayed, however, a largely prevailing form with an assessed molecular mass of 13.0 kDa, which points to a dimeric structure. Kinetic characterization of protein p2 showed a broad pH optimum in the range 6.7–7.6 using yeast RNA as substrate; also, it was shown that activity was unaffected by EDTA, Mg2+ and phosphate. The enzyme did not accept as substrate any homopolyribonucleotide, which points to a rather narrow substrate specificity. This was also confirmed by incubating p2 with tRNAfMetMet (fMet, N‐formylmethionine) from Escherichia coli: the hydrolysis products were thus identified as 3′‐phosphooligonucleotides.
Isolation and characterization of an endogenous Na+,K+ ‐ATPase‐specific inhibitor from pig urine
Authors: TAMURA Masaaki , HARRIS Thomas M. , KONISHI Fumiko , INAGAMI Tadashi
Published: March 2005

A Na+,K+‐ATPase inhibitor was purified from 88.6 I pig urine with a yield of approximately 10 μg. It inhibits the ouabain‐sensitive uptake of 86Rb into human erythrocytes and the specific binding of ouabain to Na+,K+‐ATPase. It also exhibits cross‐reactivity to anti‐ouabain serum. The purification procedure consisted of adsorption chromatography on an Amberlite XAD‐2 column, preparative scale C18 low‐pressure liquid chromatography (LPLC), and five steps of HPLC with five different types of reverse‐phase columns. The dose dependence of the purified substance for the inhibition of ouabain‐sensitive Na+,K+‐ATPase activity and 86Rb uptake into human erythrocytes, and for the ouabain‐displacing activity, paralleled those of ouabain, spanning two orders of magnitude in concentration range. However, the curve obtained from the cross‐immunoreactivity with antiouabain serum did not parallel that of ouabain. The inhibitory potencies of the purified substance against the Na+ ‐pump and ouabain‐binding were diminished with increasing K+ concentration, exhibiting characteristics typical of cardiac glycosides. This substance had no effect on Ca2+ ‐ATPase activity in human erythrocyte plasma membrane and skeletal‐muscle sarcoplasmic reticulum, nor on Mg2+ ‐ATPase activity. Acid treatment with 6 M HCl at 115°C for less than 1 min destroyed approximately 82% of the inhibitory activity of the purified substance against Na+ ‐pump activity. Alkaline treatment with 0.2 M NaOH at 23°C for 2 h and heat treatment at 150°C for 30 min partially destroyed the inhibitory activity. Boiling for 10 min and digestion by various enzymes did not affect the activity. Molecular mass was estimated to be 620 Da by gel‐filtration column chromatography. Preliminary MS analysis suggested that the purified substance has a molecular mass of 625 Da. An 1H‐NMR study revealed that this substance does not contain a tertiary methyl group. The results suggest that the purified Na+,K+ ‐ATPase inhibitor is not a peptide and is distinct from any of the known cardiotonic steroids or various substances previously reported to exhibit Na+,K+ ‐ATPase inhibitory activity. Thus, the purified substance may be a novel endogenous regulator of Na+,K+ ‐ATPase.
Complete amino acid sequence of the regulatory light chain of obliquely striated muscle myosin from earthworm, Lumbricus terrestris
Authors: SERWE Maria , MEYER Helmut E. , CRAIG Anthony G. , CARLHOFF Dietrich , D'HAESE Jochen
Published: March 2005

Amino acid sequence analysis of the regulatory light chain of obliquely striated muscle myosin from earthworm, Lumbricus terrestris, was performed completely. The polypeptide consists of 195 amino acids with a calculated molecular mass of 21 943 Da. From the arrangement of amino acid residues, the first EF‐hand domain appears to be a specific Ca2+ ‐binding site. The unusually long N‐terminal region of about 40 amino acids, which is characterized by accumulation of basic amino acids, is similar to that of the myosin A1 catalytic light chain from rabbit skeletal muscle.
Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli
Authors: ARCONE Rosaria , ARPAIA Giuseppina , RUOPPOLO Margherita , MALORNI Antonio , PUCCI Piero , MARINO Gennaro , IALENTI Armando , DI ROSA Massimo , CILIBERTO Gennaro
Published: March 2005

Lipocortin or annexin 1 is a calcium‐dependent phospholipid‐binding protein which probably acts as a glucocorticoid‐ regulated anti‐inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio‐β‐D‐galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion‐exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N‐terminal alanine; tryptic peptide mapping by fast‐atom‐bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin‐induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose‐related response is observed.
Evidence of serine‐protease activity closely associated with Drosophila alcohol dehydrogenase
Authors: FIBLA Joan , ATRIAN Sílvia , GONZÀLEZ‐DUARTE Roser
Published: March 2005

With the use of monoclonal antibodies against alcohol dehydrogenase (ADH) we detected ADH proteolysis in different Drosophila melanogaster tissues during development [Visa, N., Fibla, J., SantaCruz, M. C. & Gonzàlez‐Duarte, R. (1992) J. Histochem. Cytochem. 40, 39–49]. We now report the analysis of this proteolytic activity in crude homogenates and in purified ADH preparations of several Drosophila species. Our results indicate that in non‐denaturing IEF gels the proteolytic activity comigrates with native ADH electromorphs of all the species analyzed. In addition, we show that it copurifies with ADH and is responsible for the instability of apparently homogeneous ADH preparations in the presence of SDS. When purified ADH preparations were analyzed, the endogenous proteolytic activity yielded the same banding pattern as that obtained with crude homogenates. Even after rechromatography on Sephacryl S‐200, the usual last step in our standard purification protocol, the proteolytic activity remained associated with the ADH fractions. Among the various agents which could explain the ADH‐linked proteolytic effect, a pre‐existing nicked state of the enzyme or chemical proteolysis have been ruled out. The kinetics observed on pure ADH preparations, the effect of specific protease inhibitors and substrate specificity have led us to ascribe this activity to the subtilase serine‐protease family. Given that proteolysis is evident even in rechromatographed Sephacryl S‐200 fractions, if incubated in SDS for enough time, we propose two alternative hypotheses to explain this phenomenon. First, the proteolytic activity may come from a protease which is inseparable from the ADH active forms and second, the ADH itself may behave as a subtilase when it adopts a particular conformation. Moreover, the previously reported differential banding pattern during development suggests a role for this activity in vivo, in which fatty acids could produce the inducer effect attributed to SDS in vitro.
Fast myosin heavy chain diversity in skeletal muscles of the rabbit: heavy chain IId, not IIb predominates
Authors: AIGNER Sigrid , GOHLSCH Bärbel , HÄMÄLÄINEN Nina , STARON Robert S. , UBER Andreas , WEHRLE Ulrike , PETTE Dirk
Published: March 2005

The myosin heavy chain (HC) composition of various rabbit muscles was analysed at both the mRNA and the protein level. S1‐nuclease mapping was performed with a cDNA probe specific for myosin HCIIa, yielding a fully protected sequence for HCIIa, a partially protected sequence for HCIIb, and an additional signal putatively assigned to HCIId. At the protein level, three fast myosin HC isoforms, HCIIa, HCIIb and HCIId, were separated by gradient PAGE. The results obtained at the protein level were in agreement with the findings at the mRNA level. The expression of appreciable amounts of myosin HCIIb, the predominating isoform of fast‐twitch muscles in rat and mouse, was restricted in the rabbit to only a few muscles, i.e. adductor magnus, gastrocnemius, latissimus dorsi and vastus lateralis. Typical fast‐twitch muscles such as extensor digitorum longus, tibialis anterior and psoas contained only minute amounts of HCIIb. The HCIId isoform, demonstrated in the present study for the first time in rabbit, is the predominating fast myosin HC isoform in this species. Electrophoretic analyses of myosin HC in histochemically defined single fibers confirmed the lack of fibers expressing only HCIIb in tibialis anterior, whereas such fibers were found in the adductor magnus. In addition to fiber types IIB, IID, and IIA expressing HCIIb HCIId, and HCIIa, respectively, an appreciable amount of hybrid fibers coexpressing two HC isoforms at various ratios were found: HCIIb > HCIId; HCIId > HCIIb; HCIId > HCIIa; HCIIa > HCIId; HCIIa > HCI; HCI > HCIIa. This fiber‐type spectrum indicates possible fiber‐type transitions in the order IIB ↫ IIBD ↫ IIDB ↫ IID ↫ IIDA ↫ IIAD ↫ IIA ↫ IIC ↫ IC ↫ I.
Homology of pyridoxal‐5′‐phosphate‐dependent aminotransferases with the cobC (cobalamin synthesis), nifS (nitrogen fixation), pabC (p‐aminobenzoate synthesis) and malY (abolishing endogenous induction of the maltose system) gene products
Authors: MEHTA Perdeep K. , CHRISTEN Philipp
Published: March 2005

Bacterial deletion mutants have indicated that the gene products of cobC, nifS, pabC and malY participate in important metabolic pathways, i.e. cobalamin synthesis, nitrogen fixation, synthesis of p‐aminobenzoate and the regulation of the maltose system, respectively. However, the proteins themselves and their specific functions have not yet been identified. In the course of our studies on the evolutionary relationships among aminotransferases, we have found that the above gene products are homologous to aminotransferases. Profile analysis [Gribskov, M., Lüthy, R. & Eisenberg, D. (1990) Methods Enzymol. 183, 146–159] based on the amino acid sequences of certain subgroups of aminotransferases as probes attributed significant Z scores in the range 5–20 SD to the deduced amino acid sequences of the above gene products as included in the protein data base. Reciprocal profile analyses confirmed the homologies. All known aminotransferases are pyridoxal‐5′‐phosphate‐dependent enzymes and catalyze the reversible transfer of amino groups from amino acids to oxo acids. The sequence homologies suggest that the above gene products are aminotransferases or other closely related pyridoxal‐5′‐phosphate‐dependent enzymes probably catalyzing transformations of amino acids involving cleavage of a bond at Cα.
Cloning and Expression of a Xenopus Liver cDNA Encoding a Fructose‐Phosphate‐Insensitive Regulatory Protein of Glucokinase
Authors: Veiga‐Da‐Cunha Maria , Detheux Michel , Watelet Nathalie , Van Schaftingen Emile
Published: March 2005

Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to fructose 6‐phosphate and fructose 1‐phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551–556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619‐amino‐acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349–381) and in the 70 carboxy terminal‐residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to fructose 6‐phosphate, fructose 1‐phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose‐phosphate‐insensitive protein of lower vertebrates is homologous to the fructose‐6‐phosphate‐sensitive and fructose ‐1‐phosphate‐sensitive protein found in mammals.
Purification and Characterization of S‐adenosyl‐l‐methionine:norcoclaurine 6‐O‐Methyltransferase from Cultured Coptis japonica Cells
Authors: Sato Fumihiko , Tsujita Tetsuya , Katagiri Yasuyuki , Yoshida Shigeru , Yamada Yasuyuki
Published: March 2005

S‐adenosyl‐l‐methionine:norcoclaurine 6‐O‐methyltransferase (norcoclaurine 6‐O‐methyltransferase), which catalyzes the transfer of the S‐methyl group of S‐adenosyl‐l‐methionine to the 6‐hydroxyl group of 1,2,3,4‐tetrahydro‐1‐[(4‐hydroxyphenyl)methyl]‐6,7‐isoquinolinediol (norcoclaurine), was purified from cultured Coptis japonica cells and its enzymic properties were characterized. Purified norcoclaurine 6‐O‐methyltransferase had apparent pI 4.7, a native molecular mass of 95 kDa (determined by gel filtration) and subunit molecular mass of 40 kDa (SDS/PAGE). The enzyme did not require a divalent cation for activity, and the addition of Fe2+, Cu2+, Co2+, Zn2+, Mn2+, or Ni2+ at 5 mM severely inhibited enzyme activity. Neither p‐chloromercuribenzoate, N‐methylmaleimide nor iodoacetamide inhibited enzyme activity at 1 mM. 5, 6‐Dihydro‐9, 10‐dime‐thoxybenzo[g]‐1,3‐benzodioxolo[5,6‐a]quinolizinium (berberine, the end‐product of the biosynthetic pathway in which norcoclaurine 6‐O‐methyltransferase catalyzes an intermediate step) also inhibited the activity by 50% at 10 mM. Norcoclaurine 6‐O‐methyltransferase methylated both (S)‐norcoclaurine and (R)‐norcoclaurine and (R,S)‐norlaudanosoline. Further characterization of substrate‐saturation kinetics and product inhibition of the purified enzyme indicated that norcoclaurine 6‐O‐methyltransferase follows a bi‐bi ping‐pong mechanism with Km, values of 2.23 mM and 3.95 mM for (R,S)‐norlaudanosoline and S‐adenosyl‐l‐methionine, respectively, while Ki values for S‐adenosyl‐homocysteine versus S‐adenosyl‐l‐methionine and (R,S)‐norlaudanosoline were 2.1 mM and 0.18 mM, respectively.
High‐resolution Electron Microscopic Studies on the Quaternary Structure of Ornithine Aminotransferase from Pig Kidney
Authors: Lünsdorf Heinrich , Hecht Hans‐Jürgen , Tsai Hsin
Published: March 2005

The ornithine aminotransferase (OAT) from pig kidney has been studied on the basis of high‐resolution electron microscopy and the morphological appearance of the apoenizyme and holoenzyme have been examined. The quaternary structure of the OAT molecules in the presence of 5 mM pyridoxal 5′‐phosphate could be established. The enzyme molecule appears to be built up of two morphological units, called M1. The native holoenzyme, termed morphological unit M2, measures 10.9 nm in length and 5.8 nm in width and its molecular mass is approximately 168 kDa, based on electron microscopical calculations. Since the enzyme is composed of only one type of 45‐kDa subunit, the holoenzyme is a homotetramer. Each M1 is composed of two subunits and, as seen in top‐view projection, has an oval to triangular shape. Upon tilting to 40° the triangular shape changes into three distinct centers of mass. This morphological differentiation reflects the inner organization of M1, i.e. the shape of the individual subunit deviates from strictly globular proteins. This observation is compatible with the notion that the 45‐kDa subunit consists of one large and one small domain. By tilting to 40°, both large domains in M1 represent two of the three centers of mass, while the third center of mass is attributed to the superposition of both small domains. Thus, the four domains of both subunits in M1, in accordance with the triangular top‐view projection, are quasi‐tetrahedrally arranged. Since the change in shape of M1 upon tilting is only obvious in one of the two halves of the native OAT, it suggests that both morphological units of M2 are oriented asymmetrically relative to one another.
Importance of General Regulatory Factors Rap1p, Abf1p and Reb1p for the Activation of Yeast Fatty Acid Synthase Genes FAS1 and FAS2
Authors: Schüller Hans‐Joachim , Schütz Armin , Knab Sabine , Hoffmann Brigitte , Schweizer Eckhart
Published: March 2005

The fatty acid synthase genes FAS1 and FAS2 of the yeast Saccharomyces cerevisiae are under transcriptional control of pathway‐specific regulators of phospholipid biosynthesis. However, site‐directed mutagenesis of the respective cis ‐acting elements upstream of FAS1 and FAS2 revealed that additional sequences activating both genes must exist. A deletion analysis of the FAS1 promoter lacking the previously characterized inositol/choline‐responsive‐element motif defined a region (nucleotides –760 to –850) responsible for most of the remaining activation potency. Gel‐retardation experiments and in‐vitro DNase footprint studies proved the binding of the general regulatory factors Rap1p, Abf1p and Reb1p to this FAS1 upstream region. Mutation of the respective binding sites led to a drop of gene activation to 8% of the wild‐type level. Similarly, we also demonstrated the presence of a Reb1p‐binding site upstream of FAS2 and its importance for gene activation. Thus, in addition to the previously characterized FAS‐binding factor 1 interacting with the inositol/choline‐responsive‐element motif, a second motif common to the promoter regions of both FAS genes could be identified. Transcription of yeast fatty acid synthase genes is therefore subjected to both the pathway‐specific control affecting genes of phospholipid biosynthesis and to the activation by general transcription factors allowing a sufficiently high level of constitutive gene expression.
The Extracellular Domain of the Epidermal Growth Factor Receptor
Authors: Brown Pamela M. , Debanne Maria T. , Grothe Suzanne , Bergsma Derk , Caron Mireille , Kay Cyril , O'Connor‐Mccourt Maureen D.
Published: March 2005

The binding of epidermal growth factor (EGF) or an EGF‐like growth factor to the EGF receptor is the initial event which leads to receptor activation, and consequently the induction of cell growth. In order to study this binding interaction in detail, we produced the extracellular domain of the EGF receptor (EGFR) using the baculovirus expression system. Affinity‐labeling and Western‐blot analyses revealed that the baculovirus‐infected insect cells secrete active EGFR extracellular domain relatively efficiently, however a significant amount of inactive EGFR extracellular domain is retained within the cells. The apparent dissociation constant (Kd)of the secreted EGFR extracellular domain for EGF and transforming growth factor α (TGF‐α), as determined using an immobilized receptor binding assay, was approximately 200 nM. Interestingly, this Kd value is 30–40‐fold lower than that of the full‐length EGFR derived from detergent‐solubilized A431 cell membranes. The stoichiometry of binding of the EGFR extracellular domain to EGF and TGF‐α was examined by band‐shift analysis on non‐denaturing PAGE and was estimated to be 1:1. We have also shown, using sedimentation equilibrium analysis, that ligand binding induces significant dimerization of the EGFR extracellular domain. Finally, we carried out site‐specific mutagenesis on the EGFR extracellular domain in order to define the ligand‐binding region. We identified amino acid residues which are close to the binding site since they are common to the epitopes of several ligand‐competitive monoclonal antibodies. However, these residues do not contribute directly to ligand binding since the affinity of the mutated EGFR extracellular domain for EGF and TGF‐α was unaffected.
Peptide Substrate Specificity and Properties of the zinc‐endopeptidase Activity of Botulinum Type B Neurotoxin
Authors: Shone Clifford C. , Roberts April K.
Published: March 2005

Clostridium botulinum type B neurotoxin has been shown to be a zinc endopeptidase specific for vesicle‐associated membrane protein (VAMP). A synthetic peptide of human/rat VAMP‐2 [VAMP‐2‐(60–94)] is cleaved by the neurotoxin with the same specificity as that demonstrated for the membrane associated protein (at the Gln76‐Phe77 bond) and has been used to study the properties of the endopeptidase activity of the neurotoxin. Cleavage of the VAMP‐2 peptide was demonstrated by both botulinum type B neurotoxin (Km= 3.3×10−4M) and by its purified light subunit (Km= 3.5×10−4M). The endopeptidase displayed a pH optimum of 7.0–7.5 and was inhibited by greater than 0.2 M NaCl and greater than 0.05 M sodium phosphate. Neurotoxin which had been inactivated by dialysis against EDTA could be reactivated by incubation with various divalent cations, notably Zn2+ and Cu2+. The substrate specificity of botulinum type B neurotoxin was studied using various analogues of VAMP‐2 (60–94). The neurotoxin cleaved selectively to the N‐terminal side of phenylalanine and tyrosine; no activity was observed with either leucine, valine or alanine in the P′1 position. The properties of the P1 amino acid were less critical; the neurotoxin cleaving the C‐terminus of glutamine, asparagine and alanine. A substrate analogue with valine in the P1 position corresponding to the sequence of rat VAMP‐1 was not cleaved. The rate of cleavage of a substrate analogue representing the sequence of human VAMP‐1, however, was more than twofold that of the VAMP‐2 peptide. The properties and substrate specificity of botulinum type B neurotoxin suggest that the toxin represents a novel class of endopeptidase which requires a specific peptide substrate conformation for the expression of proteolytic activity.
Geometrical and Conformational Properties of Ganglioside GalNAc‐GD1a, IV4GalNAcIV3Neu5AcII3Neu5AcGgOse4Cer
Authors: Acquotti Domenico , Cantu Laura , Ragg Enzio , Sonnino Sandro
Published: March 2005

The aggregative properties of GalNAc‐GD1a, ganglioside, in comparison with those of GD1a, have been investigated and correlated to the intrinsic conformation and mobility of the oligosaccharide chain of the molecules. Micellar parameters in aqueous solution (molecular mass, hydrodynamic radius as well as the surface area at the lipid/water interface and the packing parameter of the monomer inserted in the aggregate) are measured by the laser light‐scattering technique. The presence of a further GalNAc residue causes a 22% increase in molecular mass, contrary to expectation. Oligosaccharide moiety three‐dimensional structures have been modeled using molecular mechanics and dynamics calculations, based on NOE interactions observed for native gangliosides dissolved in deuterated dimethylsulfoxide or, as mixed micelles with fully deuterated dodecylphosphocholine, in D2O. Compared with GD1a, the GalNAc‐GD1a, is less mobile, thus influencing the surface area, this lower mobility together with the GalNAc‐GD1a, conformation leads to a larger number of monomers participating in the formation of the micelle. The results further substantiate the model in which the three‐dimensional structure and the intrinsic dynamic properties of the oligosaccharide chain affect the geometrical properties of the aggregate.
Characterization of the Human Gene (TBXAS1) Encoding Thromboxane Synthase
Authors: Miyata Atsuro , Yokoyama Chieko , Ihara Hayato , Bandoh Setsuko , Takeda Osamu , Takahashi Ei‐ichi , Tanabe Tadashi
Published: March 2005

The gene encoding human thromboxane synthase (TBXAS1) was isolated from a human EMBL3 genomic library using human platelet thromboxane synthase cDNA as a probe. Nucleotide sequencing revealed that the human thromboxane synthase gene spans more than 75 kb and consists of 13 exons and 12 introns, of which the splice donor and acceptor sites conform to the GT/AG rule. The exon‐intron boundaries of the thromboxane synthase gene were similar to those of the human cytochrome P450 nifedipine oxidase gene (CYP3A4) except for introns 9 and 10, although the primary sequences of these enzymes exhibited 35.8% identity each other. The 1.2‐kb of the 5′‐flanking region sequence contained potential binding sites for several transcription factors (AP‐1, AP‐2, GATA‐1, CCAAT box, xenobiotic‐response element, PEA‐3, LF‐A1, myb, basic transcription element and CAMP‐response element). Primer‐extension analysis indicated the multiple transcription‐start sites, and the major start site was identified as an adenine residue located 142 bases upstream of the translation‐initiation site. However, neither a typical TATA box nor a typical CAAT box is found within the 100‐b upstream of the translation‐initiation site. Southern‐blot analysis revealed the presence of one copy of the thromboxane synthase gene per haploid genome. Furthermore, a fluorescence in situ hybridization study revealed that the human gene for thromboxane synthase is localized to band q33‐q34 of the long arm of chromosome 7. A tissue‐distribution study demonstrated that thromboxane synthase mRNA is widely expressed in human tissues and is particularly abundant in peripheral blood leukocyte, spleen, lung and liver. The low but significant levels of mRNA were observed in kidney, placenta and thymus.
Axial Coordination and Reduction Potentials of the Sixteen Hemes in High‐Molecular‐Mass Cytochrome c from Desulfovibrio Vulgaris (Hildenborough)
Authors: Verhagen Marc F. J. M. , Pierik Antonio J. , Wolbert Ronnie B. G. , Mallée Leonard F. , Voorhorst Wilfried G. B. , Hagen Wilfred R.
Published: March 2005

A spectroelectrochemical study is described of the sixteen hemes in the high‐molecular‐mass, monomeric cytochrome c (Hmc) from the periplasmic space of Desulfovibrio vulgaris, strain Hildenborough. One of the hemes has special properties. In the oxidized state at pH 7 it is predominantly high‐spin, S= 5/2, with a g⊥ value of less than 6 indicative of quantum‐mechanical mixing with a low‐lying (800 cm−1) S= 3/2 state; the balance is probably a low‐spin derivative. The high‐spin heme has an Em,7.5 value of +61 mV. The fifteen other hemes are low‐spin bis‐histidine coordinated with Em,7.5 values of approximately –0.20 V. Two of these hemes exhibit very anisotropic EPR spectra with a g1 value of 3.65 characteristic for strained bis‐histidine coordination. A previous proposal, namely that methionine is coordinated to one of the hemes [Pollock, W. B. R., Loufti, M. Bruschi, M. Rapp‐Giles, B. J., Wall, J. & Voordouw, G. (1991) J. Bacteriol. 173, 220] is disproved using spectroscopic evidence. Contrasting electrochemical data sets from two previous studies [Tan, J. & Cowan, J. A. (1990) Biochemistry 29, 4886; Bruschi, M., Bertrand, P., More, C., Leroy, G., Bonicel, J., Haladjian, J., Chottard, G., Pollock, W. B. R. & Voordouw, G. (1992) Biochemistry 31, 3281] are not consistent with our EPR titration results and are not reproducible. Hmc can be reduced by D. vulgaris Fe‐hydrogenase in the presence of molecular hydrogen.
Isolation and 1H‐NMR Spectroscopic Identification of Poly(3‐Hydroxybutanoate) from Prokaryotic and Eukaryotic Organisms
Authors: Seebach Dieter , Brunner Andreas , Bürger H. Michael , Schneider Jakob , Reusch Rosetta N.
Published: March 2005

Trace amounts of poly[(R)‐3‐hydroxybutanoate] were isolated from competent Escherichia coli, spinach, bovine serum albumin, beef heart mitochondria, and aortal tissues, all sources in which it is not accumulated as storage material. Its identity was in all cases proved by 1H‐NMR spectroscopy. In some runs, the poly[(R)‐3‐hydroxybutanoate] isolated from competent E. coli also contained some 3‐hydroxyvalerate, an observation confirmed by 1H‐NMR spectroscopy and gas chromatography. The absolute configuration of the polymers isolated from E. coli and spinach was shown to be (all‐R) by gas chromatography on chiral columns.
Functional Reconstitution of ATP‐Dependent Transporters from the Solubilized Hepatocyte Canalicular Membrane
Authors: Büchler Markus , Böhme Matthias , Ortlepp Helga , Keppler Dietrich
Published: March 2005

The hepatocyte canalicular membrane contains several primary‐active ATP‐dependent export carriers including one for bile salts and one for leukotriene C4 and related conjugates. The molecular identity of both transporters has not been fully elucidated. To establish a transport assay that allows the purification and identification of the proteins involved in ATP‐dependent bile salt transport and in leukotriene C4 transport, we reconstituted solubilized hepatocyte canalicular membranes into phospholipid bilayers using a rapid dilution method. The proteoliposomes formed exhibited both [3H]taurocholate and [3H]leukotriene C4 uptake, which was much higher in the presence of ATP than in the presence of the non‐hydrolyzable ATP‐analog AdoPP [CH2]P or in the absence of nucleotides. Nucleotide requirement and osmotic sensitivity of [3H]taurocholate transport indicates true transport into the vesicle lumen. Optimized conditions for reconstitution included the addition of a high concentration of an osmolyte (glycerol) and the presence of exogenous phospholipids (0.3%) during solubilization. Highest transport rates were obtained by reconstitution into acetone/ether‐precipitated Escherichia coli phospholipid supplemented with 20% cholesterol and by use of octylglucoside concentrations between 30 mM and 50 mM. Taurocholate transport was non‐competitively inhibited by vanadate (Ki= 39 μM). The kinetic parameters of cyclosporin A inhibition (Ki= 2.6 μM for taurocholate and 4.3 μM for leukotriene C4 transport) as well as the affinities of taurocholate (Km= 12 μM) and leukotriene C4 (Km= 0.5 μM) in the proteoliposome system indicate that the reconstitution resulted in functionally active transport systems, which are representative of ATP‐dependent transport in the intact plasma membrane.