bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification, Characterization and Partial Peptide Microsequencing of Progesterone 5β‐Reductase from Shoot Cultures of Digitalis purpurea
Authors: Gärtner Dorothea E. , Keilholz Wieland , Seitz Hanns Ulrich
Published: March 2005

Progesterone 5β‐reductase, which catalyzes the reduction of progesterone to 5βT‐pregnane‐3,20‐dione, was purified 770‐fold to homogeneity from the cytosolic fraction of shoot cultures of Digitalis purpurea. This purification involved DEAE‐Sephacel, affinity chromatography (Blue‐Sepharose CL‐6B and adenosine 2′,5′‐bisphosphate‐Sepharose 4B) and elution from a gel matrix after non‐dissociating PAGE. The molecular mass determined by SDS/PAGE was 43 kDa and the molecular mass determined by gel‐filtration chromatography on calibrated Sephadex G‐200 was 280 kDa, thus indicating that the native protein is a polymer consisting of several subunits. The purified enzyme had a Km value of 6 μM for NADPH and 34 μM for progesterone. The enzyme had a strong substrate specificity for progesterone. The relative rates for other steroids such as testosterone, cortisone and cortisol were much lower. The trypsin digestion of the purified progesterone 5β‐reductase resulted in 100 peptide fragments. The largest fragment after trypsin digestion and sequence analysis consisted of 13 amino acids.
Relationships Between Molecular Interactions (Nucleotides, Lipids and Proteins) and Structural Features of the Bovine Brain 21‐kDa Protein
Authors: Bucquoy Sophie , Jollès Pierre , Schoentgen Françoise
Published: March 2005

A cytosolic 21‐kDa protein isolated from bovine brain was demonstrated to bind hydrophobic ligands, particularly phosphatidylethanolamine. It was encountered in several tissues and species; however, its accurate function remained partially unknown. In order to obtain information from its structural features, we built a molecular model which revealed it to possess a nucleotide‐binding site. In the present research, we describe the affinity of the bovine brain 21‐kDa protein for nucleotides, and its association with cytosolic proteins, small GTP‐binding proteins and lipid droplets. Our results suggest that, through its association with small GTP‐binding proteins, the 21‐kDa protein is implicated in signal mechanisms during cell growth and maturation.
The DNA‐Binding Efficiency of Sp1 is Affected by Redox Changes
Authors: Ammendola Rosario , Mesuraca Maria , Russo Tommaso , Cimino Filiberto
Published: March 2005

We have previously demonstrated that the DNA‐binding efficiency of Sp1 is greatly decreased in nuclear extracts from 30‐month‐old rat tissues compared to those from young ones, although its gene appears to be normally expressed. As reactive oxygen intermediates are known to accumulate in aged animals, we investigated the effect of oxidation on the Sp1 DNA‐binding activity. Electrophoretic mobility shift assays and DNase I footprintings showed that high concentrations of dithiothreitol, added to the aged tissue extracts, fully restore the Sp1 DNA‐binding efficiency. However, in young nuclear extracts hydrogen peroxide treatment strongly decreases the Sp1 DNA‐binding activity that is restored by the treatment with high dithiothreitol concentrations. To ascertain whether the oxidative stress is directed toward the Sp1 molecule alone, or whether it acts on unknown Sp1 cofactor(s) necessary for DNA binding, we purified Sp1 from young rat liver and demonstrated that when the purified protein is added to aged nuclear extracts, it efficiently binds to its DNA cis‐element. Moreover, purified Sp1 treated with hydrogen peroxide lost its ability to bind its cis‐element and the DNA‐binding efficiency was fully restored after incubation with dithiothreitol.
Overexpression of the myristoylated alanine‐rich C‐kinase substrate in Rat1 cells increases sensitivity to calmodulin antagonists
Authors: Herget Thomas , Broad Simon , Rozengurt Enrique
Published: March 2005

The acidic 80‐kDa myristoylated alanine‐rich C‐kinase substrate protein (80‐kDa MARCKS) is the major protein‐kinase‐C substrate in rodent fibroblasts. To elucidate its function, we transfected the cDNA coding for the 80‐kDa MARCKS protein into Rat1 fibroblasts. One clone, called Rat1–80K, expressed 4.5 ± 0.8–fold and 9.5 ± 1.5‐fold higher levels of 80‐kDa MARCKS protein under quiescent and growing conditions, respectively, compared to mock or untransfected control cells. Southern‐blot and Northern‐blot analyses of Rat1–80K showed intact integration and correct transcription of the introduced 80‐kDa MARCKS gene. The overexpressed 80‐kDa MARCKS protein was phosphorylated and translocated from the membrane to the cytoplasmic fraction. Since 80‐kDa MARCKS has been described as a calmodulin‐binding protein in in vitro studies, we investigated the effects of the calmodulin antagonists N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide and triflouperazine on the entry into the S‐phase of the cell cycle in intact cells. DMA synthesis by Rat1–80K cells was more sensitive to either N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide or triflouperazine than that of control cells. Our results suggest that Overexpression of the 80‐kDa MARCKS protein reduces the free concentration of calmodulin in the cell.
Aspartate‐Bond Isomerization Affects the Major Conformations of Synthetic Peptides
Authors: Szendrei Gyorgyi I. , Fabian Heinz , Mantsch Henry H. , Lovas Sandor , Nyéki Olga , Schön Istvan , Otvos Laszlo
Published: March 2005

The aspartic acid bond changes to an β‐aspartate bond frequently as a side‐reaction during peptide synthesis and often as a post‐translational modification of proteins. The formation of β‐aspartate bonds is reported to play a major role not only in protein metabolism, activation and deactivation, but also in pathological processes such as deposition of the neuritic plaques of Alzheimer's disease. Recently, we reported how conformational changes following the aspartic‐acid‐bond isomerization may help the selective aggregation and retention of the amyloid β peptide in affected brains (Fabian et al., 1994). In the current study we used circular dichroism, Fourier‐transform infrared spectroscopy, and molecular modeling to characterize the general effect of the β aspartate‐bond formation on the conformation of five sets of synthetic model peptides. Each of the non‐modified, parent peptides has one of the major secondary structures as the dominant spectro‐scopically determined conformation: a type I β turn, a type II β turn, short segments of α or 310 helices, or extended β strands. We found that both types of turn structures are stabilized by the aspartic acid‐bond isomerization. The isomerization at a terminal position did not affect the helix propensity, but placing it in mid‐chain broke both the helix and the β‐pleated sheet with the formation of reverse turns. The alteration of the geometry of the lowest energy reverse turn was also supported by molecular dynamics calculations. The tendency of the aspartic acid‐bond isomerization to stabilize turns is very similar to the effect of incorporating sugars into synthetic peptides and suggests a common feature of these post‐translational modifications in defining the secondary structure of protein fragments.
Identification and Biosynthesis of N1,N9‐Bis(Glutathionyl)Aminopropylcadaverine (Homotrypanothione) in Trypanosoma Cruzi
Authors: Hunter Karl J. , Quesne Sarah A. Le , Fairlamb Alan H.
Published: March 2005

Radiolabelling studies using tritiated omithine, arginine and lysine, together with the relevant amino acid decarboxylase enzyme assays, indicate that the epimastigote stage of Trypanosoma cruzi is unable to synthesise significant amounts of putrescine and cadaverine de novo, compared to the amounts of these diamines scavenged from the growth medium. Radiolabelled putrescine is readily incorporated into spermidine, spennine and the trypanosomatid‐specific polyamine‐glutathione conjugate trypanothione (N1, N8‐bis(glutathionyl)spermidine). Likewise, radiolabelled cadaverine is incorporated into the analogous polyamines aminopropylcadaverine, bis(aminopropyl)cadaverine and another major unidentified component. Subsequent studies showed this major component to be a novel polyamine‐thiol conjugate whose structure was confirmed by chemical synthesis to be N1,N9‐bis(glutathionyl)aminopropylcadaverine (homotrypanothione). Kinetic analyses using recombinant T. cruzi trypanothione reductase demonstrated that homotrypanothione disulphide is readily reduced by this enzyme with kinetic parameters similar to trypanothione disulphide, suggesting that it is a physiological substrate in vivo. Thus the epimastigote form of T. cruzi differs significantly from the African trypanosomes and Leishmania in (a) being unable to synthesise significant amounts of diamines de novo, (b) converting significant amounts of putrescine and cadaverine to spermine and bis(aminopropyl)cadaverine, respectively and (c) the ability to synthesise homotrypanothione as well as trypanothione. The implications of these findings with respect to the prospective chemotherapy of Chagas’ disease are discussed.
Expression of properdin in human monocytes
Authors: SCHWAEBLE Wilhelm , HUEMER Hartwig P. , MÖST Johannes , DIERICH Manfred P. , STRÖBEL Marion , CLAUS Christine , REID Kenneth B. M. , ZIEGLER‐HEITBROCK H. W. Löms
Published: March 2005

Properdin is the only known positive regulator of the alternative pathway of complement activation. Northern blot analysis of cell lines derived from fibroblasts, B‐cells, hepatoma cells, and cells of the monocyte‐macrophage lineage revealed properdin expression only in the myelomonocytic cell line HL‐60, in the monoblastic cell line U‐937 and in the monocytic line Mono Mac 6. Culture of Mono Mac 6 cells for 24 h with phorbol 12‐myristate 13‐acetate, bacterial lipopolysaccharide and the cytokines interleukin‐1β and tumour necrosis factor‐α enhanced mRNA abundance, with the strongest effect (tenfold) being observed with the lipopolysaccharide. In contrast, recombinant interferon‐γ consistently halved properdin mRNA abundance. The same pattern was found for the secretion of properdin as detected by ELISA of Mono Mac 6 supernatants. The suppressive effect of interferon‐γ on properdin mRNA abundance was also demonstrated for primary blood monocytes. The data suggest that the expression and secretion of this complement regulatory protein by monocytes is differentially regulated by cytokines and link the immune response with alternative pathway activation.
Selective processing of submandibular rat 1 protein at dibasic cleavage sites
Authors: ROUGEOT Catherine , ROSINSKI‐CHUPIN Isabelle , NJAMKEPO Elisabeth , ROUGEON François
Published: March 2005

The amino acid sequence of submandibular rat 1 (SMR1) protein, deduced from its cDNA sequence, led to the prediction that the SMR1 gene encodes a hormone‐like precursor [Rosinski‐Chupin, I., Tronik, D. & Rougeon, F. (1988) Proc. Natl Acad. Sci. USA 85, 8553–8557]. SMR1 contains an N‐terminal putative secretory signal sequence and a tetrapeptide (QHNP), located between dibasic amino acids which constitute the most common signal for prohormone processing.

We have isolated and characterized from the male rat submandibular gland and its secretions three structurally related peptides, namely an undecapeptide (VRGPRRQHNPR), a hexapeptide (RQHNPR) and a pentapeptide (QHNPR) generated from SMR1 by selective proteolytic cleavages at pairs of arginine residues.

The biosynthesis of these peptides is subjected to distinct regulatory pathways depending on the organ, sex and age of the rat. Furthermore, the peptides are differentially distributed in the submandibular gland and in resting or epinephrine‐elicited submandibular salivary secretions, suggesting distinct proteolytic pathways for their maturation. The undecapeptide is generated in the gland of both male and female rats, but under basal conditions it is only released into the saliva in male animals. The hexapeptide is produced in large amounts in the gland of adult male rats and released into the saliva in both resting and stimulated conditions. The pentapeptide appears only in the male saliva and is present mostly under stimulated conditions. In addition, administration of epinephrine induces the release of the hexapeptide from the submandibular gland into the bloodstream. The evidence indicates that the rat submandibular gland can function as a dual exocrine and endocrine organ for the SMR1‐derived hexapeptide, as has been reported for nerve growth factor, epidermal growth factor, renin and kallikrein.

Although the biological activities of the SMR1‐derived peptides are not yet known, their high production and adrenergic‐induced release only into the saliva and bloodstream of adult male rats, suggest a physiological involvement in some male‐specific processes.
Mammalian phosphatidylinositol 3′‐kinase induces a lethal phenotype on expression in Schizosaccharomyces pombe; comparison with the VPS34 gene product
Authors: KODAKI Tsutomu , WOSCHOLSKI Rüdiger , EMR Scott , WATERFIELD Michael D. , NURSE Paul , PARKER Peter J.
Published: March 2005

The 110‐kDa catalytic subunit of the phosphatidylinositol 3′‐kinase (p110) is shown to be expressed in Schizosaccharomyces pombe as a functional protein, as judged by the accumulation of 3′‐phosphorylated lipids in vivo and the extraction of 3′‐kinase activity in vitro. On expression of p110, the cells fail to grow and lose viability. In contrast, while the Saccharomyces cerevisiae protein Vps34p can be expressed in S. pombe as a functional, extractable, phosphatidylinositol 3‐kinase, expression of this protein fails to increase substantially 3′‐phosphorylated lipids in vivo and does not induce a phenotype equivalent to that induced by p110. The results indicate that (over‐)‐accumulation of 3′‐phosphorylated inositol lipids in S. pombe causes loss of viability.
Overexpression of a cytochrome P‐450 of the 2a family (Cyp2a‐5) in chemically induced hepatomas from female mice
Authors: JOUNAÏDI Youssef , BONFILS Claude , PÉRIN François , NEGISHI Masahiko , LANGE Reinhard
Published: March 2005

Chemical hepatocarcinogenesis in female mice, induced by 5,9‐dimethyl(7H)dibenzo[c,g]carbazole, leads to the overexpression of a cytochrome P‐450 of the 2a family. This protein was identified as Cyp2a‐5, by the use of immunoblots obtained from isoelectric focusing gels. This method allowed the distinction of Cyp2a‐5 from Cyp2a‐4, another mouse liver cytochrome P‐450, by taking advantage of their slightly different pI values. The theoretical pI values, determined from the amino acid sequence, were pI 9.91 for Cyp2a‐4 and pI 10.01 for Cyp2a‐5. Other structurally related forms were not detected. In hepatomas from female mice, only the Cyp2a‐5 form was overexpressed (2–3 fold). Male mice showed a weak expression of Cyp2a‐4 and Cyp2a‐5 in control liver samples and in hepatomas. The expression of both forms was increased more than fivefold upon castration. Pyrazole induces specifically the Cyp2a‐5 form. The Cyp2a‐5 overexpression was correlated with enhanced microsomal coumarin‐7‐hydroxylase and testosterone‐15α‐hydroxylase activities.

An immunohistochemical study showed that Cyp2a‐4 and Cyp2a‐5 are expressed uniformly in female livers, but centrilobularly in male livers. In hepatomas, this localisation is perturbed; in females we observed a focal cell localisation, and the Cyp2a‐containing cells were often hypertrophic and polyploid. In hepatomas from male mice, the Cyp2a‐containing cells became dispersed.

From a comparison with other studies, the Cyp2a‐5 overexpression appears to be a general feature of hepatocarcinogenesis in mice.
Identificatión of a glutamate residue at the active site of xylanase A from Schizophyllum commune
Authors: BRAY Mark R. , CLARKE Anthony J.
Published: March 2005

The xylanase A (endo‐1,4‐β‐d‐xylan xylanhydrolase) of the basidiomycete Schizophyllum commune was treated with the powerful carboxylate‐modifying reagent 1‐(4‐azonia‐4,4—dimethylpentyl)‐3‐ethylcarbodiimide iodide (EAC) in the presence of substrate. This treatment was followed by complete inactivation of the enzyme with [14C]EAC after the removal of excess reagent and protecting ligand. The inactivated enzyme was digested with endoproteinase Arg‐C or trypsin, and peptides were separated and purified using reverse‐phase high‐performance liquid chromatography. Following sub‐digestion of individual radioactive peptides with staphylococcal V8 protease and endoproteinase Lys‐C, amino acid composition analysis and sequencing analysis revealed that the [14C]EAC label was bound exclusively to Glu87. Comparisons of the primary sequences of related xylanases with that of xylanase A revealed that Glu87 is a highly conserved residue. Based on this similarity and the mechanism of carbodiimide action, Glu87 is proposed to act as the nucleophile in the catalytic mechanism of xylanase A. The possible environment of the putative catalytic glutamate residue was explored using hydrophobic‐cluster analysis and secondary‐structure prediction based on the primary sequence of xylanase.
Structure of the sea urchin hatching enzyme gene
Authors: GHIGLIONE Christian , LHOMOND Guy , LEPAGE Thierry , GACHE Christian
Published: March 2005

The sea urchin embryo develops from an encased to a free‐living larva by secreting at an early stage the hatching enzyme, a metalloprotease which hydrolyses a protective envelope derived from the egg extracellular matrix. Genomic clones containing the entire hatching enzyme gene were isolated from a λ phage sea urchin library and the complete sequence of the transcription unit was determined. The hatching enzyme gene spans 6.3 kb and comprises 9 exons. The exon/intron organization of the hatching enzyme gene is similar but not identical to those of the vertebrate collagenases and stromelysins. The position and/or phase of several introns are different even in the N‐terminal moiety where similarity between echinoderm and vertebrate enzymes was first detected. The active‐center domain is encoded by a 1–1 class exon whose sequence, length and borders are highly conserved and might be considered as coding for a protein module. Adjacent to the activecenter exon, the hatching enzyme gene has an additional 1–1 exon which codes for a threonine‐rich region. This provides further evidence that the matrix‐degrading metalloproteinases evolved by shuffling exons of the 1–1 class. Phylogeny analysis indicates a close relationship between the sea urchin and vertebrate enzymes.
Backbone dynamics of (1–71)bacterioopsin studied by two‐dimensional 1H‐15N NMR spectroscopy
Authors: OREKHOV Vladislav Yu. , PERVUSHIN Konstantine V. , ARSENIEV Alexander S.
Published: March 2005

The backbone dynamics of a uniformly 15N‐labelled proteolytic fragment (residues 1–71) of bacteriorhodopsin, solubilized in two media [methanol/chloroform (1:1), 0.1 M 2HCO2NH4 and SDS micelles] have been investigated using two‐dimensional proton‐detected heteronuclear 1H‐15N NMR spectroscopy. A set of longitudinal and transverse relaxation rates of 15N nuclei and 1H‐15N NOE were obtained for 61 backbone amide groups. The contribution of the conformational exchange to transverse relaxation rates of individual nitrogens was elucidated using a set of different rates of the Carr‐Purcell‐Meiboom‐Gill (CPMG) spin‐lock pulse train. We found that most of the backbone amide groups are involved in the co‐operative exchange process over the rate range 103–104s−1, with the chemical‐shift dispersion near 1 ppm. Contributions of conformational exchange to the measured transverse relaxation were essentially suppressed by the 3‐kHz (spin‐echo period τ= 0.083 ms) CPMG spin‐lock. Under these conditions, the measured longitudinal, transverse relaxation rates and NOE values were interpreted using the model‐free approach of Lipari and Szabo [Lipari, G. & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546–4559]. In both media used, the protein exhibits very similar dynamic properties, and has overall rotational correlation times of 7.0 ns and 6.6 ns in organic mixture and in SDS micelles, respectively. In addition to overall rotation of the molecule, the backbone N‐H vectors are involved in two types of internal motions; fast, on a time scale of <20 ps, and intermediate, close to 1 ns. Distinctly mobile regions are identified by a large decrease in the overall order parameter and correspond to N‐terminal residues (residues 1–7 both for organic solvent and micelles), C‐terminal residues (residues 65–71 and 69–71 for organic solvent and micelles, respectively) and residues connecting α helices (residues 33–41 and 33–38, for organic solvent and micelles, respectively). A decrease in the order parameter was also observed for residues next to Pro50, indicating a higher flexibility in this region. Thus, backbone dynamic parameters of (1–71)bacterioopsin are in good correspondence with its spatial structure [Pervushin, K. V., Orekhov, V. Yu., Popov, A., Musina, L. Yu., Arseniev, A. S., (1994) Eur. J. Biochem., in the press]. The observed conformational exchange behavior of α helices seems to be induced by the flickering helix‐helix interaction and could be important for the functioning of bacteriorhodopsin.
Study of the interaction between salivary proline‐rich proteins and a polyphenol by 1H‐NMR spectroscopy
Authors: MURRAY Nicola J. , WILLIAMSON Michael P. , LILLEY Terence H. , HASLAM Edwin
Published: March 2005

The interaction between salivary proline‐rich proteins and plant polyphenols (tannins) in the oral cavity and their subsequent precipitation influences the taste, texture and nutritional value of food; it is thought to be responsible for the astringency of many foods and beverages. To investigate the interaction, two‐dimensional 1H‐NMR studies have been carried out on the binding of a representative polyphenol, pentagalloyl glucose, to two synthetic peptides (19 and 22 residues in length) that are typical of the repeat sequence of mouse salivary proline‐rich protein MP5. Intermolecular nuclear Overhauser effects and chemical shift changes show that the main binding sites on the peptides are proline residues together with the preceding amide bond and amino acid. The interaction is principally a hydrophobic association between a galloyl ring and the pyrrolidine ring face containing the Cα proton, but secondary hydrogen‐bonding effects help to stabilise the complex. Very similar interactions are seen for both peptides. The conformation of the peptides remains extended on binding. The chemical shift changes seen for many of the peptide protons can be fitted to a simple binding curve with dissociation constant of around 40 mM, but some protons show evidence of cooperative binding involving several galloyl groups. Higher concentrations of pentagalloyl glucose lead to a reduced off‐rate from the complex and eventual precipitation which implies that precipitation is caused by a kinetic competition between aggregation and dissociation of the complex.
The glucose transporter of Escherichia coli Assignment of the 1H, 13C and 15N resonances and identification of the secondary structure of the soluble IIB domain
Authors: GOLIC GRDADOLNIK Simona , EBERSTADT Matthias , GEMMECKER Gerd , KESSLER Horst , BUHR Andreas , ERNI Bernhard
Published: March 2005

The IICBGlc subunit of the Escherichia coli glucose transporter consists of two domains, the membrane‐spanning IIC domain, and the hydrophilic IIB domain which contains the phosphorylation site (Cys421). A functional form of the IIB domain was over‐expressed separately and isotopically labelled with 13C and 15N. A variety of 15N‐edited and 13C, 15N triple‐resonance NMR experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances. Based on the evaluation of conformationally sensitive parameters including NOE effects, scalar couplings and chemical shifts, the secondary structure of the IIB domain is presented. The protein is comprised of four β‐strands forming an antiparallel β‐sheet, two larger α‐helices at the N‐ and C‐termini and a smaller helical structure of residues 52–58.
Evolutionary relationships among pyridoxal‐5′‐phosphate‐dependent enzymes
Authors: ALEXANDER Frederick W. , SANDMEIER Erika , MEHTA Perdeep K. , CHRISTEN Philipp
Published: March 2005

Pyridoxal‐5′‐phosphate‐dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio‐specificity.

In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the imine linkage with the coenzyme. This family was thus named α family. It comprises glycine hydroxymethyltransferase, glycine C‐acetyltransferase, 5‐aminolevulinate synthase, 8‐amino‐7‐oxononanoate synthase, all aminotransferases (with the possible exception of subgroup III), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol‐lyase which, however, catalyze β‐elimination reactions. The β family includes L‐ and D‐serine dehydratase, threonine dehydratase, the β subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze β‐replacement or β‐elimination reactions. The γ family incorporates O‐succinylhomoserine (thiol)‐lyase, O‐acetylhomoserine (thiol)‐lyase, and cystathionine γ‐lyase, which catalyze γ‐replacement or γ‐elimination reactions, as well as cystathionine β‐lyase.

The α and γ family might be distantly related with one another, but are clearly not homologous with the β family. Apparently, the primordial pyridoxal‐5′‐phosphate‐dependent enzymes were regio‐specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal‐5′‐phosphate‐dependent enzymes seem to be unrelated with the α, β or γ family by the criterion of profile analysis: alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B6 enzymes.
Expression cloning of an equine T‐lymphocyte glycoprotein CD2 cDNA
Authors: TAVERNOR Angela S. , KYDD Julia H. , BODIAN Dale L. , JONES E. Yvonne , STUART David I. , DAVIS Simon J. , BUTCHER Geoffrey W.
Published: March 2005

An equine CD2 cDNA has been isolated by monoclonal antibody screening of a T‐lymphocyte cDNA library. The cDNA contained an open reading frame of 1041 bp encoding a translated product of 347 amino acids. Northern blotting analysis revealed a single mRNA species expressed in spleen, thymus and activated peripheral lymphocytes. The predicted amino acid sequence has 50–65% identity with the human, rat and mouse CD2 sequences with greatest similarity shared with the human homologue. Evolutionarily conserved structural and functional domains in CD2 were identified by comparing the sequences of the equine, human, mouse and rat CD2 homologues in the context of the recently derived crystal structure of rat soluble CD2 [Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K. & Stuart, D. I. (1992) Nature 360, 232–239]. The key conserved features of the extracellular region included core residues necessary to preserve the structural integrity of the molecule, residues in the linker region likely to maintain the unique domain organization of CD2, an array of highly charged residues in the putative ligand‐binding face of the molecule and glycosylation‐signal distributions that render the putative ligand‐binding GFCC'C” face of domain 1 relatively unhindered by glycosylation.
Aluminium fluoride and magnesium, activators of heterotrimeric GTP‐binding proteins, affect high‐affinity binding of the fungal toxin fusicoccin to the fusicoccin‐binding protein in oat root plasma membranes
Authors: DE BOER Albertus H. , VAN DER MOLEN Gineke W. , PRINS Hidde B. A. , KORTHOUT Henrie A. A. J. , VAN DER HOEVEN Paulus C. J.
Published: March 2005

The fusicoccin‐binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high‐affinity [3H]fusicoccin binding to almost zero in a concentration‐dependent way, with an optimum at approximately 20 mM sodium fluoride. The presence of magnesium (> 100 μM) was required for the inhibitory action of fluoride, whereas addition of low amounts of aluminium (25 μM) shifted the fluoride optimum to lower concentrations. Fluoride changes the biochemical properties of the binding protein in a reversible manner, because the inhibition was both prevented and reversed by 1 M ammonium sulphate.

The combined effects of aluminium, fluoride and magnesium are reminiscent of the action of activated GTP‐binding proteins. Since no functional assay for GTP‐binding‐protein activity in plants is available yet, GTP‐binding‐protein activation by fluoride and magnesium was deduced from competition with binding of [γ‐35S]GTP[S] to purified plasma membranes. Indeed, fluoride (20 mM) completely blocked the specific binding of [γ‐35S]GTP[S].

It is concluded that the inhibitory effect of fluoride upon the binding of fusicoccin is indirect and mediated through activated GTP‐binding proteins. A hypothesis on the mechanism of fusicoccin action is presented wherein the fusicoccin‐binding protein is one component of a signal‐transduction chain, two or more steps downstream of a heterotrimeric GTP‐binding protein.
Electropermeabilization mediates a stable insertion of glycophorin A with Chinese hamster ovary cell membranes
Authors: EL OUAGARI Khalid , BENOIST Hervé , SIXOU Sophie , TEISSIE Justin
Published: March 2005

Electropulsation allowed us to incorporate glycophorin A, an integral membrane protein, into mammalian nucleated cell membranes (Chinese hamster ovary cells). The induction of stable protein association is effective only when the field intensity is higher than its threshold value, creating membrane permeabilization to small molecules. Under controlled conditions, cell viability was only slightly altered by this treatment. Pulse number and duration controlled both the number of modified cells and incorporated molecules. The phenomena was temperature dependent. An average of 5X104 molecules/cell was bound. About 80% of cells in the pulsed population were observed to incorporate glycophorin. The protein incorporation was shown to be stable 48 h after electroassociation. Electrically bound proteins were shared between the cells after each division. As enhanced binding is detected if glycophorin is added after the pulses, it is the long‐lived alteration of the membrane mediated by the pulses which supports the association.
The metabolism of 2‐fluoro‐2‐deoxy‐d‐glucose in human chondrocytes and its incorporation into keratan sulfate proteoglycans
Authors: FEDDERS Gönna , KOCK Rüdiger , VAN DE LEUR Eddy , GREILING Helmut
Published: March 2005

The incorporation of 2‐fluoro‐2‐deoxy‐d‐[14C]glucose in proteoglycans was investigated in a cell culture system, where human articular chondrocytes were cultured in high‐cell‐density thin‐layer soft agarose. The proteoglycans were solubilized from the culture medium and the cell layer fraction by extracting with a guanidine hydrochloride buffer and purified by an ion‐exchange‐chromatography (DEAE‐Sepharose CL‐6B). With enzymic decomposition experiments concerning the glycosaminoglycan side‐chains it could be shown that 65–69% were digestible by keratanase, whereas 21–29% of the 14C‐labeled proteoglycans were digested with chondroitinase AC/ABC. The main constituent of the 2‐fluoro‐2‐deoxy‐d‐[14C]glucose‐metabolites present in the glycosaminoglycan side chains of the proteoglycans was 2‐fluoro‐2‐deoxy‐d‐[14C]galactose. Therefore, 2‐fluoro‐2‐deoxy‐d‐glucose was preferentially incorporated into keratan sulfate.

We investigated the effect of non‐radioactive 2‐fluoro‐2‐deoxy‐d‐glucose on UDP‐sugar and proteoglycan biosynthesis after incubation periods of 1–30 h. A high 2‐fluoro‐2‐deoxy‐d‐glucose concentration in the culture medium did not influence the pool size of UDP‐N‐acetylhexosamines, but UDP‐d‐glucose, UDP‐d‐galactose, UDP‐d‐glucuronic acid, UDP‐2‐fluoro‐2‐deoxy‐d‐glucose, UDP‐2‐fluoro‐2‐deoxy‐d‐galactose and UDP‐2‐fluoro‐2‐deoxy‐d‐glucuronic acid accumulated in the chondrocytes time dependently. In a pulse/chase experiment the retarded synthesis of fluorinated UDP‐sugars was proved. The half‐lives (t1/2) for UDP‐2‐fluoro‐2‐deoxy‐d‐glucose and UDP‐2‐fluoro‐2‐deoxy‐d‐galactose were about 7.7 h and 13.3 h, respectively. UDP‐2‐fluoro‐2‐deoxy‐d‐glucuronic acid could be found with delay. The incubation with 2‐fluoro‐2‐deoxy‐d‐glucose and [14C]glucosamine resulted in a decreased radioactive labelling of chondroitin sulfate and keratan sulfate.
The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space
Authors: STUART Rosemary A. , GRUHLER Albrecht , van der KLEI Ida , GUIARD Bernard , KOLL Hans , NEUPERT Walter
Published: March 2005

The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted. Import and processing of precytochrome b2, (pb2), a precursor carrying a bipartite presequence, into the intermembrane space was also strongly dependent on matrix ATP. Preproteins, consisting of 220 or more residues of pb2 fused to dihydrofolate reductase, showed the same requirement for matrix ATP, whereas the import of shorter fusion proteins (up to 167 residues of pb2) was largely independent of matrix ATP. For those intermembrane‐space‐targeted proteins that did need matrix ATP, the dependence could be relieved either by unfolding these proteins prior to import or by introducing a deletion into the mature portion of the protein thereby impairing the tight folding of the cytochrome b5 domain.

These results suggest the following: (a) The import of matrix‐targeted preproteins, in addition to a membrane potential ΔΨ, requires matrix ATP [most likely to facilitate reversible binding of mitochondrial heat‐shock protein 70 (mt‐Hsp70) to incoming precursors], for two steps, securing the presequence on the matrix side of the inner membrane and for the completion of translocation; (b) in the case of intermembrane‐space‐targeted precursors with bipartite signals, the function of ATP/mt‐Hsp70 is not obligatory, as components of the intermembrane‐space‐sorting pathway may substitute for ATP/mt‐Hsp70 function (however, if a tightly folded domain is present in the precursor, ATP/mt‐Hsp70 is indispensable); (c) unfolding on the mitochondrial surface of tightly folded segments of preproteins is facilitated by matrix‐ATP/mt‐Hsp70.
pH‐dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro
Authors: SØRENSEN Susanne O. , VAN DEN HAZEL H. Bart , KIELLAND‐BRANDT Morten C. , WINTHER Jakob R.
Published: March 2005

Carboxypeptidase Y is a vacuolar enzyme from Saccharomyces cerevisiae. It enters the vacuole as a zymogen, procarboxypeptidase Y, which is immediately processed in a reaction involving two endoproteases, proteinase A and proteinase B. We have investigated the in vitro activation of purified procarboxypeptidase Y by purified proteinase A. This has identified two different processing intermediates; one active and one inactive. The intermediates define a 33 amino acid segment of the 91 amino acid propetide as sufficient for maintaining the enzyme in an inactive state. The inactive intermediate was isolated from a processing reaction at neutral pH. In order to investigate the influence of vacuolar pH on processing in vivo, the autoactivation of proteinase A and its processing of procarboxypeptidase Y were studied in a vma2 prbl mutant, which is deficient in vacuolar acidification and proteinase B activity. Efficient processing of procarboxypeptidase Y in the absence of proteinase B is dependent on acidic vacuolar pH, and the processing at neutral pH is slow and takes place in two steps similar to those identified in vitro.
ATP‐independent DNA renaturation catalyzed by a protein from Ustilago maydis
Authors: COLE Allyson D. , KMIEC Eric B.
Published: March 2005

A protein that catalyzes the renaturation of complementary strands of DNA has been purified from mitotic cells of the lower eukaryote Ustilago maydis. The most highly purified fraction contains a polypeptide with a molecular mass of 20 kDa as determined by SDS/PAGE and glycerol gradient sedimentation. DNA reannealing is enhanced by the presence of a divalent cation but does not require ATP nor any other nucleotide triphosphate. Reassociation proceeds with fast kinetics as more than 60% of the DNA is reannealed within 4 min at a 30:1 nucleotide/protein monomer ratio, results which suggest that the protein acts in a stoichiometric fashion. Amino acid analysis revealed that the protein contained an elevated level of basic residues and low levels of tryptophan and tyrosine. The protein binds to an oligonucleotide of ten residues but not to one having only five. As judged by agarose gel assays, the protein does not catalyze strand‐transfer reactions but does promote the annealing of a 58‐residue polynucleotide onto single‐stranded circles and gapped linear duplexes. These latter reactions are dependent on the presence of DNA sequence similarity between the pairing partners.
A family of hexosephosphate mutases in Saccharomyces cerevisiae
Authors: BOLES Eckhard , LIEBETRAU Wolfgang , HOFMANN Michael , ZIMMERMANN Friedrich K.
Published: March 2005

The Saccharomyces cerevisiae PGM1 and PGM2 genes encoding two phosphoglucomutase isoenzymes have been isolated and sequenced. The drived protein sequences are closely related to one another and show distinct sequence similarities to the human and rabbit phosphoglucomutases, especially in the region supposed to constitute the active site. PGM1 and PGM2 are located on chromosomes XI and XIII, respectively, just upstream of the known genes YPK1 and YKR2 coding for a pair of closely related putative protein kinases. These observations suggest that an extended region of DNA arose by the process of gene duplication. Cells deleted for both, PGM1 and PGM2, could not grow on galactose. No residual phosphoglucomutase activity could be measured in crude extracts or in permeabilized cells of pgm1/2 double mutants. Unexpectedly, growth with glucose was not impaired and mutant cells were still able to accumulate trehalose and glycogen, although at a reduced level. Two further genes could be isolated and characterized which when over‐expressed on a multi‐copy plasmid could restore growth on galactose of the pgm1/2 double deletion mutant. Multi‐copy complementation was due to a sharply increased level of phosphoglucomutase activity. Partial sequencing and characterization of the two genes revealed one of them to be SEC53 encoding phosphomannomutase. No extended sequence similarities could be found in the databases for the second gene. However, part of the derived amino acid sequence contained a region of high similarity to the active‐site consensus sequence of hexosephosphate mutases from different organism. Further investigations suggest that a complex network of mutases exist in yeast which interact and can partially substitute for each other.
Liver‐specific DNase I‐hypersensitive sites and DNA methylation pattern in the promoter region of a 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase gene
Authors: ZIMMERMANN Pascale L. N. , ROUSSEAU Guy G.
Published: March 2005

The mRNA for the liver isozyme of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase is transcribed from the L promoter of gene A. We show here that L‐promoter activity is tissue specific. To identify on the gene in situ potential cis‐acting sequences, we have examined 15 kb of its 5′ region for DNase I‐hypersensitive sites detectable on chromatin. We have also evaluated the DNA methylation status of the 3.7‐kb encompassing the L promoter. Five DNase I‐hypersensitive sites were detected on liver chromatin, three upstream (M1 at position –4500, L2 at position –1000, L1 at position –200) and two downstream (I1 at position +3000, I2 at position +3500) from the L‐type mRNA transcription initiation site. Their presence correlated with transcriptional activity as they were not observed on chromatin from kidney, a tissue where gene A is not expressed. Sites M1 and L1 Corresponded to the M and L promoters, respectively, providing in vivo evidence for a promoter localization obtained earlier with cloned DNA only. Site I2 coincided with a glucocorticoid‐responsive unit described by others, but its presence did not depend on glucocorticoids. Thus, sites L2 and I1 could correspond to novel control elements. While DNA was methylated around position –2000 both in liver and kidney, downstream from that position it was fully demethylated in liver but not in kidney. This pattern changed during development of fetal liver. The data suggest mechanisms for the lack of activity of the L promoter in kidney and for its activation in developing and adult liver.
Molecular organization at the glycoprotein‐complex‐binding site of dystrophin
Authors: SUZUKI Atsushi , YOSHIDA Mikiharu , HAYASHI Kensuke , MIZUNO Yuji , HAGIWARA Yasuko , OZAWA Eijiro
Published: March 2005

Direct interaction between the C‐terminal portion of dystrophin and dystrophin‐associated proteins was investigated. The binding of dystrophin to each protein was reconstituted by overlaying bacterially expressed dystrophin fusion proteins onto the blot membranes to which dystrophin‐associated proteins were transferred after separation by SDS/PAGE with the following results. (a) Among the components of the glycoprotein complex which links dystrophin to the sarcolemma, a 43‐kDa dystrophin‐associated glycoprotein binds directly to dystrophin. Although at least one of the binding sites of this protein resides within the cysteine‐rich domain of dystrophin, a contribution of additional amino acid residues within the first half of the C‐terminal domain was also suggested for more secure binding. (b) Two other proteins also directly bind to dystrophin. Their binding sites are suggested to reside within the last half of the C‐terminal domain which is alternatively spliced depending on the tissue type.

Previously, based on the enzyme digestion experiments, we showed that the binding site for the glycoprotein complex on dystrophin is present within the cysteine‐rich domain and the first half of the C‐terminal domain [Suzuki, A., Yoshida, M., Yamamoto, H. & Ozawa, E. (1992) FEBS Lett. 308, 154–160]. Here, we have extended this work and found that the region which is involved in interaction with the complex is widely extended to the entire length of this part of the molecule. On the basis of the present results, we propose a model of molecular architecture at the binding site for the complex on dystrophin.
Cell‐cycle‐regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and Ser38
Authors: BRATTSAND Göran , MARKLUND Ulrica , NYLANDER Karin , ROOS Göran , GULLBERG Martin
Published: March 2005

Oncoprotein 18 (Op18) has been independently identified due to its increased phosphorylation in response to external signals and its up‐regulated expression in acute leukemia. We have identified two serine residues of Op18 that are phosphorylated after triggering by the T cell antigen receptor. One of these residues, Ser25, was shown to be a likely substrate for the mitogen‐activated protein (MAP) kinase, while the other residue, Ser16, was shown to be phosphorylated in response to increased intracellular calcium. Our previous site‐mapping studies of Op18 also revealed that basal phosphorylation of Op18 is mainly located on Ser38, which was found to be the primary in vitro phosphorylation site of p13suc1‐precipitated cdc2 kinase activitie. These findings raised the possibility that Op18 may be a substrate for both receptor‐regulated calcium‐induced protein kinases and the MAP kinase family, as well as being a substrate for the cell‐cycle‐regulated cdc2 kinase family. In the present report we have performed site‐mapping studies of cell‐cycle‐regulated fluctuations of Op18 phosphorylation. The results reveal that S‐phase progression of a synchronised leukemic T cell line is associated with increased phosphorylation of both the Ser25 and Ser38 residues. Moreover, during mitosis, a burst of phosphorylation was observed and at this stage of the cell cycle a major fraction of Op18 was phosphorylated at multiple sites. Phosphorylation of Op18 during mitosis was located primarily on Ser38 and to lesser extent on Ser25, Ser16 and at an unidentified C‐terminal residue. In vitro phosphorylation experiments, employing two distinct members of the cdc2 kinase family, were consistent with involvement of both p34‐cdc2 and p33‐cdk2 in cell‐cycle‐regulated phosphorylation of Ser25 and Ser38 of Op18. Most importantly, the ratio of Ser25/Ser38 phosphorylation observed in vitro, using either p34‐cdc2 or p33‐cdk2, was found to be the same as the ratio observed in intact cells during all phases of the cell cycle. These findings suggest that Op18 may be a physiological substrate for several members of the cdc2 kinase family during both the S‐phase and the mitotic phase of the cell cycle.
Identification of a major poly‐N‐acetyllactosamine‐containing cell‐surface glycoprotein of mouse teratocarcinoma cells
Authors: SPILLMANN Dorothe , FINNE Jukka
Published: March 2005

Mouse teratocarcinoma F9 cells were induced to primitive endoderm differentiation with retinoic acid, and poly‐N‐acetyllactosamine‐containing surface glycoproteins were identified by radiolabelling endo‐β‐galactosidase‐cleavable glycans with galactosyltransferase and radiolabelled UDP‐galactose. One major radiolabelled band with an apparent size of 250–500 kDa was identified which differed from the known poly‐N‐acetyllactosamine‐containing glycoproteins laminin, fibronectin, lysosome‐associated membrane protein (LAMP)‐1 and LAMP‐2. This acidic glycoprotein, resistant to glycosaminoglycan‐degrading enzymes and proteases, was purified by extraction and phase partition with Triton X‐114, octyl Sepharose and Helix pomatia lectin chromatography. The purified glycoprotein could be digested by endo‐β‐galactosidase and glycopeptide N‐glycosidase F to an apparent size of 160–240 kDa. During retinoic‐acid‐induced differentiation into primitive endoderm cells, the glycoprotein showed a several‐fold increase and a broadening to an apparent size of 200–> 700 kDa. The glycoprotein was no longer detected in retinoic‐acid and dibutyryl‐cAMP‐treated cells which had undergone further differentiation to parietal endoderm cells, nor in the permanently differentiated parietal endoderm line F9‐AC. The results suggest that the glycoprotein is a major carrier of poly‐N‐acetyllactosamine chains on differentiating teratocarcinoma F9 cells, and that its expression as revealed by the poly‐N‐acetyllactosamine labelling method is regulated by the stage of cellular differentiation.