bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of globin mRNA, β‐globin mRNA segments and RNA homopolymers by immobilized protein of polysomal globin messenger ribonucleoprotein
Authors: GÖRLACH Matthias , HERMANN Marita , SCHWEMMLE Martin , HILSE Kurt
Published: March 2005

The binding of rabbit globin mRNA, in‐vitro‐generated β‐globin mRNA segments, and RNA homopolymers by proteins of rabbit reticulocyte polysomal messenger ribonucleoproteins (mRNP) after SDS gel electrophoresis and electroblotting was examined. The polysomal mRNP proteins have a higher affinity for mRNA than for rRNA and tRNA while having a higher affinity for polypurine than polypyrimidine homopolymers. Binding experiments with synthetic poly(A) and with segments of β‐globin mRNA transcribed from a cDNA in vitro revealed a set of polysomal mRNP proteins which preferentially bind the poly(A)‐free β‐globin mRNA. A protein of Mr 90000 binds specifically the 3′‐nontranslated trailer of the poly(A)‐free β‐globin mRNA and not the poly(A)‐containing globin mRNA. Another set of proteins preferentially binds poly(A). The latter group of proteins contains a prominent species of Mr 72000, which is most likely the rabbit poly(A)‐binding protein. Three polysomal mRNP proteins which bound rabbit globin mRNA did not bind preferentially any of the other RNA probes used.
Nuclearly inherited diuron‐resistant mutions conferring a deficiency in the NADH – or succinate – ubiquinone oxidoreductase actitiy in Saccharomyces cerevisiae
Authors: MEUNIER Brigitte , COLSON‐CORBISIER Anne‐Marie , LEMESLE‐MEUNIER Danielle
Published: March 2005

In Saccharomyces cerevisiae, diuron, antimycin and myxothiazol block the respiratory pathway at the bc1 complex level. Nuclearly inherited mutations located at the DIU3 and DIU4 loci confer in vitro resistance to diuron and cross‐resistance to antimycin and myxothiazol at the NADH oxidase level. The mutant strains do not exhibit diuron resistance at the quinol–cytochrome‐c oxidoreductase level. Thus, the apparent resistance does not seem to be the result of a modification of the inhibitory sites. Instead, the quinone reduction rate was found to be altered in the mutant. The diu3 mutations lead to a deficiency of the NADH–ubiquinone oxidoreductase activity, and the diu4 mutations to a deficiency of the succinate–ubiquinone oxidoreductase activity. On the basis of the model of Kröger and Klingenberg, a decrease of quinone reduction could explain the resistance to the bc1 complex inhibitors. Thus, the apparent resistance to the bc1 complex inhibitors was found to be due to a modification of the electron transfer kinetics.
The transitory complex between photoexcited rhodopsin and transducin
Authors: BORNANCIN Frédéric , PFISTER Claude , CHABRE Marc
Published: March 2005

In the first step of the visual transduction cascade a photoexcited rhodopsin molecule, Rret*, binds to a GDP‐carrying transducin molecule, TGDP. The R*‐T interaction causes the opening of the nucleotide site in T and catalyzes the GDP/GTP exchange by allowing the release of the GDP. We have studied the influences on this R*‐T transitory complex of the occupancies of the nucleotide site in T and the retinal site in rhodopsin. After elimination of the GDP released from the bound transducin, the complex, named Rret*‐Te (ret for retinal present, e for nucleotide site empty) remains stabilized almost indefinitely in a medium whose ionic composition is close to physiological. In this complex the bound Te retains a lasting ability to interact with GDP or GTP, and Rret* remains spectroscopically in the meta‐II state, by contrast with free Rret* which decays to opsin and free retinal. Hence the R*‐T interaction which opens the nucleotide site in T conversely blocks the retinal site in Rret*. Upon prolonged incubation in a low‐ionic‐strength medium the Rret*‐Te complex dissociates partially, but the liberated Te is then unable to rebind GDP or GTP, even in the presence of Rret*; it is probably denaturated. Upon treatment of the Rret*‐Te complex by a high concentration of hydroxylamine, the retinal can be removed from the rhodopsin. The Re‐Te complex remains stable and the complexed transducin keeps its capacity to bind GTP. TGTP then dissociates from Re. The liberated Re loses its capacity to interact with a new transducin.

These data are integrated into a discussion of the development of the cascade. We stress that affinities, i.e. dissociation equilibrium constants, are insufficient to describe the flow of reactions triggered by one Rret* molecule. It depends on a few critical rapid binding and dissociation processes, and is practically insensitive to other slow ones, hence to the values of affinities that express only the ratio of kinetics constants.

The effect of the R*‐T interaction on the retinal site in rhodopsin is analogous to the effect of the binding of a G‐protein on the apparent affinity of a receptor for its agonist.
Purification and characterization of cytochrome P‐450sca from Streptomyces carbophilus
Authors: MATSUOKA Tatsuji , MIYAKOSHI Shunichi , TANZAWA Kazuhiko , NAKAHARA Kaori , HOSOBUCHI Masahiko , SERIZAWA Nobufusa
Published: March 2005

Prayastatin sodium (CS‐514) is a tissue‐selective inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, a key enzyme in cholesterol biosynthesis. This compound is obtained by microbial hydroxylation of sodium ML‐236B (compactin) carboxylate. The soluble cytochrome P‐450 was induced by sodium ML‐236B carboxylate in Streptomyces carbophilus of Actinomycetes as detected in its cell‐free extract. This cytochrome P‐450 was designated as cytochrome P‐450sca after its origin. Cytochrome P‐450sca was purified by successive chromatography on anion‐exchange, gel filtration and hydroxyapatite columns. On hydroxyapatite cytochrome P‐450sca was further separated into minor and major peaks, designated cytochrome P‐450sca‐1 and cytochrome P‐450sca‐2, respectively. Each peak yielded a single band on sodium dodecyl sulfate/polyacrylamide gels with molecular masses of 46 ± 1 kDa. The activity hydroxylating sodium ML‐236B carboxylate to pravastatin sodium was reconstituted in the presence of an electron transport system, an NADPH‐generating system and oxygen. The Ks values of the cytochromes P‐450sca‐1 and P‐450sca‐2 for sodium ML‐236B carboxylate were 179 μM and 229 μM, respectively. The CO versus reduced difference spectra of both cytochromes P‐450 showed an absorption maximum at 448.5 nm. Their substrate difference spectra with sodium ML‐236B carboxylate showed an absorption maximum at 386 nm. Amino acid analysis indicated that cytochrome P‐450sca‐1 and P‐450sca‐1 and P‐450sca‐2 contained 46% and 47% hydrophobic residues, respectively. On Western blotting, cytochromes P‐450sca‐1 and P‐450sca‐2 were immunologically identical.
Synthesis and characterization of μ‐conotoxin IIIa
Authors: BECKER Stefan , ATHERTON Eric , GORDON Robert D.
Published: March 2005

μ‐Conotoxin IIIa, a voltage‐dependent sodium channel neurotoxin, has been synthesised using solid‐phase peptide synthesis employing 9‐fluorenylmethoxycarbonyl chemistry. After cleavage from the resin, the peptide was isolated by reverse‐phase HPLC and then the six acetamidomethyl groups were removed by treatment with mercuric acetate. The reduced product so formed was purified by reverse‐phase HPLC. Protocols were developed to optimize the oxidation of the cysteine residues to form disulphide bonds. Protocols employed using air oxidation together with 2‐mercaptoethanol were the most effective. As complete oxidation was never obtained the oxidised peptide was purified by reverse‐phase HPLC. The acitivity of our products was monitored using [3H]saxitoxin binding to eel membranes. The oxidised product was able to completely block [3H]saxitoxin binding in a competitive manner. Lineweaver‐Burke analysis of [3H]saxitoxin binding gave a Ki of 1.5 nM, IC50 was determined as 26.6 nM. It was also shown that the pure synthetic μ‐conotoxin IIIa had the same retention time on reverse‐phase HPLC as the natural conotoxin IIIa. Thus an active toxin has been synthesised that can be used to probe sodium channels.
Mobility of secondary structure units of horse‐muscle acylphosphatase
Authors: SAUDEK Vladimir , WILLIAMS Robert J. P. , STEFANI Massimo , RAMPONI Giampietro
Published: March 2005

The antigenic properties of acylphosphatase are compared with its various sequential characteristics (hydrophobicity, chemical shift of the main‐chain 1H‐NMR resonances, numbers and intensities of the nuclear Overhauser enhancements, hydrogen‐deuterium exchange and sequential arrangement of the secondary structure units). The discussion is based on the complete sequential assignment of the 1H‐NMR spectrum and the knowledge of the three‐dimensional fold of the protein obtained by NMR spectroscopy from distance geometry calculations. Regions with very different degrees of mobility can be distinguished. It is found that all major antigenic sites are located in the most mobile surface loops.
Large‐scale preparation of recombinant platelet‐derived growth factor AA secreted from recombinant baby hamster kidney cells
Authors: EICHNER Wolfram , JÄGER Volker , HERBST Detlev , HAUSER Hansjörg , HOPPE Jürgen
Published: March 2005

The short isoform of platelet‐derived growth factor A (PDGF‐A) was expressed in a mammalian host (BHK‐21 cell). A cell line was obtained that secreted up to 0.3 μg/106 cells recombinant PDGF‐A chain homodimer/day into the medium. For large‐scale production of supernatant, cells were grown either in roller bottles or in 2.5‐1 stirred tank fermenters. A simple two‐step procedure was developed to purify recombinant PDGF‐AA (rPDGF‐AA). The first step was adsorption onto porous glass and the final step was reversed‐phase high‐performance liquid chromatography. The yield was 0.2 mg/1 supernatant. A total amount of 20–30 mg pure rPDGF‐AA may be obtained from a single fermenter run. Sequence analysis showed the correct amino terminus and no internal proteolytic cleavages. The specific activity was 5 ng/ml for mouse AKR‐2B cells. [125I]rPDGF‐AA had an affinity constant of approximately 0.5 nM to these cells and 25000 binding sites were estimated/cell.
Biochemical properties of conjugates of urokinase‐type plasminogen activator with a monoclonal antibody specific for cross‐linked fibrin
Authors: DEWERCHIN Maria , LIJNEN H. Roger , Van HOEF Berthe , De COCK Frans , COLLEN Désiré
Published: March 2005

Equimolar mixtures of recombinant single chain urokinase‐type plasminogen activator (rscu‐PA) and a murine monoclonal antibody (MA‐15C5) directed against fragment‐D dimer of human cross‐linked fibrin were conjugated, using the cross‐linking agent N‐succinimidyl 3‐(2‐pyridyldithio)propionate (PySSProSu). The conjugate (rscu‐PA/MA‐15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment‐D dimer with a yield of 42 ± 15% (mean ± SD, n= 3), contained an average of 1.2 ± 0.3 IgG molecules/rscu‐PA molecule. On non‐reduced SDS/PAGE it migrated as a main band with apparent Mr of 200000.

Specific amidolytic activities expressed/mass of u‐PA were < 250 IU/mg for rscu‐PA/MA‐15C5 and rscu‐PA, 140000 ± 13000 IU/mg and 100000 ± 17000 IU/mg for their plasmin‐generated two chain derivatives rtcu‐PA/MA‐15C5 and rtcu‐PA respectively. Specific activities on fibrin plates were 100000 ± 24000 IU/mg and 130000 ± 49000 IU/mg for rscu‐PA/MA‐15C5 and rtcu‐PA/MA‐15C5 respectively, as compared to 180000 ± 15000 IU/mg for both rscu‐PA and rtcu‐PA. Activation of plasminogen with rscu‐PA/MA‐15C5 (Km= 0.37 ± 0.16 μM, k2= 0.0063 ± 0.0030 s−1) or rtcu‐PA/MA‐15C5 (Km= 19 ± 3.0 μM, k2= 2.0 ± 0.10 s−1) in purified systems followed Michaelis‐Menten kinetics with Km and k2 values comparable to those of rscu‐PA and rtcu‐PA.

In an in vitro system composed of a 125I‐fibrin‐labeled whole human plasma clot immersed in citrated human plasma, dose‐ and time‐dependent lysis was obtained; 50% lysis in 2 h required 1.4 μg/ml of rscu‐PA or 0.33 μg/ml of rtcu‐PA, but only 0.22 μg u‐PA/ml of rscu‐PA/MA‐15C5 or 0.15 μg u‐PA/ml of rtcu‐PA/MA‐15C5. Addition of purified fragment‐D dimer reversed the increased fibrinolytic potency of rscu‐PA/MA‐15C5 in a concentration‐dependent way (50% inhibition at 7.2 μg fragment‐D dimer/ml).

Thus, conjugation of u‐PA moieties with the fibrin‐specific antibody MA‐15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4‐fold for rscu‐PA and 2.2‐fold for rtcu‐PA.
Improved purification and biochemical properties of phosphatidylinositol‐specific phospholipase C from Bacillus thuringiensis
Authors: KUPKE Thomas , LECHNER Max , KAIM Georg , GÖTZ Friedrich
Published: March 2005

Monophosphatidylinositol inositol phosphohydrolase (phosphatidylinositol‐specific phospholipase C. PtdIns‐PLC, EC 3.1.4.10) has been purified from a Bacillus thuringiensis culture supernatant and from the cellular fraction of a recombinant Escherichia coli clone containing the PtdIns‐PLC gene from B. thuringiensis. The two‐step purification procedure involved ion‐exchange chromatography on DEAE‐Sepharose followed by separation on a Mono‐Q/FPLC‐column with yields of 32% and 50%, respectively. The molecular mass was determined to be 34 kDa by SDS/PAGE. The isoelectric point of the enzyme was 5.15. The amino‐terminal sequences were shown to be identical for the enzymes purified from both organisms. PtdIns‐PLC was inhibited by divalent cations using mixed micelles of Triton X‐100 and pure phosphatidylinositol. PtdIns‐PLC activity was detectable on polyacrylamide gels by activity staining on phosphatidylinostiol‐containing agarose.
Catabolic pathway of oligosaccharide‐diphospho‐dolichol
Authors: CACAN René , LEPERS Anne , BELARD Michel , VERBERT André
Published: March 2005

The degradation of oligosaccharide‐diphospho‐dolichol leads to the release of oligosaccharide material ranging from (Glc)3(Man)9(GlcNAc)2‐P to (Man)3 species and further smaller species. The subcellular location of the glucosidases and mannosidases involved in this catabolic process has been investigated on the basis of their differential sensitivity towards specific inhibitors (castanospermine, deoxymannojirimycin and swainsonine): The results indicate that the first steps of degradation down to the (Man)6 species occurs in the rough endoplasmic reticulum. This result is supported by the fact that the (Man)6 species is the end product when lipid‐intermediate‐derived glucosylated oligosaccharides are incubated with purified rough endoplasmic reticulum membranes.

Swainsonine and lysosomotropic agents (chloroquine and ammonium chloride) do not affect the degradation process, thus indicating that neither Golgi apparatus nor lysosomes are involved in this catabolism. The observation of the same degradation pattern of the released oligosaccharide material in mannosidosis fibroblasts, lacking lysosomal mannosidases, confirms these results.

Finally, the subcellular distribution of the released oligosaccharide material indicates that the oligomannosides larger than (Man)6 species are sequestered in the particulate fraction whereas, in contrast, oligomannosides smaller than (Man)6 species are found predominantly in the cytosol.

Taken altogether, the experiments demonstrate that the first steps of the degradation of oligosaccharide‐diphospho‐dolichol occurs in the rough endoplasmic reticulum producing oligomannosides of the (Man)6 species which are then translocated to the cytoplasm to be further degraded.
The heparin and pentosan polysulfate binding sites of human antithrombin overlap but are not identical
Authors: SUN Xiao‐Jian , CHANG Jui‐Yoa
Published: March 2005

Four sulfated polysaccharides (unfractionated heparin, low‐molecular‐mass heparin, heparan sulfate and pentosan polysulfate) were investigated for their abilities (a) to bind antithrombin, (b) to induce conformational change of the inhibitor and (c) to potentiate antithrombin inhibition of thrombin. The binding capacity was reflected by the shielding of the heparin binding site. This was characterized by the extent to which a polysaccharide could protect chemical modification of Lys‐125 and Lys‐136, two lysyl residues of antithrombin which have been implicated in heparin binding. The conformational change was measured by fluorescence enhancement and the increased accessibility of Lys‐236 to chemical modification. Our results reveal that the events of polysaccharide binding, conformational change and the enhancement of inhibitory activity are not quantitatively interlinked. Compared to the unfractionated heparin on an equal mass basis, the low‐molecular‐mass heparin (molecular mass 4–6 kDa) binds more strongly to antithrombin, induces a greater conformational change (about twofold), but is less potent in accelerating the inhibitory activity. Both heparin and heparan sulfate shield Lys‐125 and Lys‐136 and induce a conformational change that leads to exposure of Lys‐236 and an increased fluorescence. On the other hand, pentosan polysulfate protects only Lys‐125 and causes no appreciable conformational change, although it is also capable of enhancing the antithrombin—thrombin interaction. These data clearly demonstrate that the heparin and pentosan polysulfate binding sites of antithrombin overlap (at Lys‐125) but are not identical.
Biosynthesis of 1‐alkenes in higher plants: stereochemical implications
Authors: GÖRGEN Günther , BOLAND Wilhelm
Published: March 2005

Odd numbered 1‐alkenes, such as 1‐pentadecene or 1,8,11,14‐heptadecatetraene are formed from palmitic or linolenic acid by fragmenlative decarboxylation. Incubation studies with germinating safflower (carthamus tinctorius) and (2R,3R)‐12‐phenyl[2,3‐2H2]dodecanoic acid, (2S,3S)‐12‐phenyl[2,3‐2H2]dodecanoic acid, (2R)‐12‐phenyl[2‐2H]dodecanoic acid and (2S)‐12‐phenyl[2‐2H]dodecanoic acid instead of the natural α‐linolenic acid precursor revealed the fragmentation to be an overall anti elimination of the 3‐pro(S) hydrogen and the carboxyl group (anti‐periplanar transition state geometry). Externally offered 3‐hydroxy acids are not fragmented to 1‐alkenes. The most probable mechanistic alternatives are in agreement with abstraction of the 3‐pro(S) hydrogen as a radical followed by electron transfer and fragmentation, or transient insertion of oxygen into the 3‐pro(S) C–H bond and subsequent fragmentation into an 1‐alkene and CO2 after appropriate activation. The mechanism seems to be of general importance for the biosynthesis of vinylic substructures of natural products from oxygenated precursors.
Production of human adrenocorticotropin by cleavage of alkaline‐phosphatase‐derived fusion proteins containing repetitive recognition sequences for collagenases
Authors: DAUM Joachim , DONNER Peter , GEILEN Wilhelm , HÜBNER‐KOSNEY Gisela , ISERNHAGEN Marina , SCHEIDECKER Harald , SELIGER Hartmut , BOIDOL Werner , SIEWERT Gerhard
Published: March 2005

Recombinant plasmids coding for fusion proteins which consist of human adrenocorticotropin joined to N‐terminal sequences of Escherichia coli alkaline phosphatase via collgenase‐sensitive linkers were constructed and used for the production of these proteins by transformed E. coli cells. It was shown that repetitive linkers of the form ‐Gly‐(Pro‐Xaa‐Gly)n‐Pro‐ with n≥2 were cleaved by clostridiopeptidase A (Clostridium histolyticum) by orders of magnitude faster than corresponding nonrepetitive sequences (n= 1). The C‐terminal cleavage product was Gly‐Pro‐adrenocorticotropin which could be converted to the authentic hormone by dipeptidyl peptidase IV. On the basis of these enzymatic reactions a procedure for the preparation of pure adrenocorticotropin was developed. Derivatives of alkaline phosphatase containing similar repetitive linker sequences were cleaved by clostridiopeptidase A as efficiently as the adrenocorticotropin fusion proteins.
Recognition of specific Physarumα‐tubulin isotypes by a monoclonal antibody
Authors: WALDEN Paul D. , BLINDT Adrian B. , BIRKETT Christopher R. , COX Robert A. , GULL Keith
Published: March 2005

The monoclonal antibody 6‐11B‐1 recognises specifically the acetylated form of α‐tubulin. The acetylation event occurs on a unique lysine residue, lysine 40. Using 6‐11B‐1, acetylated α‐tubulin was detected in myxamoebae but not plasmodia of Physarum polycephalum. Following chemical acetylation plasmodial α‐tubulin was detected by 6‐11B‐1. The monoclonal antibody KMP‐1 recognises certain Physarumα‐tubulin isotypes but only in nonacetylated form. Whilst recognising all the non‐acetylated fraction of myxamoebal α‐tubulin only a proportion of plasmodial α‐tubulin was recognised by KMP‐1. Peptides were synthesised corresponding to the acetylation domains (containing lysine 40) of myxamoebal α‐tubulin and the inferred acetylation domains of two plasmodial‐specific α‐tubulin isotypes. The only difference between the two peptides was at a single residue corresponding to amino acid 44 in the polypeptide. Tyrosine was present in myxamoebal α‐tubulin and glycine was present in the plasmodial specific peptides; the peptides are referred to as the Tyr44 and Gly44 peptides respectively. Both peptides in acetylated form blocked 6‐11B‐1 reactivity towards acetylated myxamoebal α‐tubulin. The Tyr44 but not the Gly44 peptide blocked KMP‐1 reactivity towards non‐acetylated myxamoebal α‐tubulin. Tyrosine at position 44 is not found in any other known α‐tubulin. Thus a unique antigenic determinant exists in certain Physarumα‐tubulin isotypes, close to the acetylation site at lysine 40. This antigenic determinant forms part of the KMP‐1 recognition epitope and explains the unique isotype selectivity of this monoclonal antibody.
Purification and characterization of angiotensin‐binding protein from porcine liver cytosolic fraction
Authors: HAGIWARA Hiromi , SUGIURA Naoaki , WAKITA Ken‐ichi , HIROSE Shigehisa
Published: March 2005

A protein that binds angiotensins with high affinity was found in porcine liver cytosol, purified to apparent homogeneity and characterized. The protein was named soluble angiotensin‐binding protein (sABP) to distinguish it from angiotensin II receptors present on plasma membranes. Purification of the protein was achieved by a combination of ammonium sulfate fractionation, hydrophobic chromatography, ion‐exchange chromatography, hydroxylapatite column chromatography and Mono Q ion‐exchange chromatography. Specific angiotensin‐binding activity, as measured using 125I‐angiotensin II, was enriched more than 3400‐fold. SDS/polyacrylamide gel electrophoresis of the purified sABP yielded a single 75‐kDa protein band, in good agreement with the molecular mass estimated by affinity labeling. sABP was very similar to the angiotensin II receptor in its sensitivity to reducing agents and in its affinities for angiotensin analogues ([Sar1, Ala8]angiotensin II > angiotensin III > angiotensin II > angiotensin I), suggesting a possible similarity between the ligand‐binding sites of sABP and the angiotensin II receptor. To obtain a clue to its physiological role(s), we examined the tissue distribution of sABP and found that this protein is widely distributed not only in the peripheral organs but also in the brain.
Purification and properties of Saccharomyces cerevisiae acetolactate synthase from recombinant Escherichia coli
Authors: POULSEN Charlotte , STOUGAARD Peter
Published: March 2005

The yeast ilv2 gene, encoding acetolactate synthase, was subcloned in an Escherichia coli expression vector. Although a major part of the acetolactate synthase synthesized by E. coli cells harbouring this vector was packaged into protein inclusion bodies, we used these recombinant E. coli cells to produce large quantities of the yeast enzyme. The yeast acetolactate synthase was purified to homogeneity using first streptomycin and ammonium sulfate precipitations, followed by T‐gel thiophilic interaction, Sephacryl S‐300 gel filtration, Mono Q anion exchange, and Superose 12 gel filtration chromatography. SDS/PAGE and gel filtration of the purified enzyme showed that it is a dimer composed of two subunits, each with the molecular mass of 75 kDa. The purified yeast acetolactate synthase was further characterized with respect to pH optimum, dependence of the substrate, pyruvate, and requirements of the cofactors, thiamin diphosphate, Mg2+, and FAD
Characterization of a membrane‐associated phosphotyrosyl protein phosphatase from the A431 human epidermoid carcinoma cell line
Authors: BÜTLER Martin T. , ZIEMIECKI Andrew , GRONER Bernd , FRIIS Robert R.
Published: March 2005

The A431 human epidermoid carcinoma cell line exhibits a 30‐100‐fold overexpression of the epidermal growth factor (EGF) receptor. We have characterized a membrane‐associated phosphotyrosyl‐protein phosphatase (PTPase) in these cells since it seemed reasonable that overexpression of the EGF‐receptor tyrosine kinase will be matched by high PTPase activity. Indeed, of 12 cell lines tested, the A431 cells had the highest specific PTPase activity. About 70% of the total cellular PTPase activity was found associated with membranes after cell fractionation. The membrane‐associated PTPase was hydrophobic as judged by its behaviour in Triton X‐114 phase partitioning. High‐performance liquid chromatography (HPLC) on a DEAE column revealed a single, homogeneous species of membrane‐associated PTPase with an apparent molecular mass of 43 kDa as determined by HPLC on a gel permeation column in the presence of Triton X‐100. Comparison of this PTPase with the membrane‐associated PTPase activities present in rat spleen and in the human chronic myelogenous leukemia cell line K562 revealed additional species resolvable by DEAE‐HPLC. These findings suggest that cells may possess different PTPase activites depending on their growth and differentiation states.
Determination of the active‐site serine of 6‐aminohexanoate‐dimer hydrolase
Authors: NEGORO Seiji , MITAMURA Toshihide , OKA Kaneo , KANAGAWA Kazuo , OKADA Hirosuke
Published: March 2005

Diisopropylfluorophosphate, an inhibitor of serine proteinase, was used to label 6‐aminohexanoate‐dimer hydrolase, a nylon oligomer degradative enzyme of Flavobacterium sp. KI72. More than 95% of the enzyme activity was lost upon incorporation of 1–1.5 molecules inhibitor/subunit of the enzyme. The tryptic peptide of the labeled enzyme was purified by HPLC (reverse‐phase partition) and its amino acid sequence was identified. Radioactivity was found to be incorporated into an 8‐amino‐acid peptide (108His‐Leu‐Leu‐Met‐Ser‐Val‐Ser‐Lys115). Amino acid alteration from Ser to Ala at the position 112 by site‐directed mutagenesis caused loss of enzyme activity to below the detection threshold (1% of the activity of the parental enzyme). These results indicate that Ser112 is essential for the activity.
Hydrodynamic, structural and magnetic properties of Megasphaera elsdenii Fe hydrogenase reinvestigated
Authors: FILIPIAK Marian , HAGEN Wilfred R. , VEEGER Cees
Published: March 2005

Megasphaera elsdenii hydrogenase has been purified to homogeneity using an FPLC procedure as the final step. The protein gives a single band in SDS/PAGE with an apparent molecular mass of 57–59 kDa. There is no second hydrogenase activity in the soluble fraction of M. elsdenii. The hydrodynamics of the enzyme have been compared to those of the two‐subunit Fe hydrogenase from Desulfovibrio vulgaris (Hildenborough) in the analytical ultracentrifuge using the absorption of the intrinsic iron‐sulfur clusters as the monitor. Sedimentation‐velocity experiments indicate the M. elsdenii enzyme (s20,w= 4.95 S) to be essentially globular, while the D. vulgaris enzyme (s20,w= 4.1 S) has a less symmetric shape. From the sedimentation equilibrium measurements under a variety of conditions an average molecular mass is calculated of 58 kDa (M. elsdenii) and 54 kDa (D. vulgaris), respectively. Pure, maximally active M. elsdenii hydrogenase has A405/A280= 0.36 and has a specific H2‐production activity of 400 μmol H2· min−1· (mg protein)−1 at 30°C and pH 8.0. The enzyme contains some 13–18 iron and acid‐labile sulfur ions/58‐kDa monomer. Eight of these Fe‐S are present as two electron‐transferring ferredoxin‐like cubanes with Em≳–0.3 V, as indicated by pH‐dependent EPR spectroscopy on the H2‐reduced enzyme. In the (re)oxidized state the remainder iron gives rise to a single S= 1/2 rhombic EPR signal. Hydrogen‐production activity, content of remainder iron and rhombic EPR signal intensity are mutually correlated. Purified hydrogenase appears to exist as a mixture of fully active holoenzyme and inactive protein still carrying the two cubanes but deficient in active‐site iron.
Tryptophan phosphorescence as a monitor of the structural role of metal ions in alkaline phosphatase
Authors: CIONI Patrizia , PIRAS Luciana , STRAMBINI Giovanni Battista
Published: March 2005

The phosphorescence properties of Trp109 in alkaline phosphatase from Escherichia coli have been utilized to probe the conformation of the polypeptide following the removal of metal ions, reconstitution with Zn2+ and Cd2+ and phosphorylation. The complete removal of metal ions induces a drastic loosening of the protein structure that extends to the inner core of the macromolecule. While binding of a single metal ion/subunit (A‐site occupancy) restores the holoconformer, practically no structuring effect is observed upon B‐site occupancy by the second incoming metal ion. An exception to this rule occurs at alkaline pH and when the adjacent subunit in the dimer is metal‐free. Under these circumstances a conformation of the subunit more compact than that of the fully saturated dimer manifests some degree of communication across the subunit interface. The binding of more than two metal ions/monomer generally destabilizes the protein, the effect being more pronounced at acid pH. Finally, the binding of inorganic phosphate restores the native‐like configuration abolishing any destabilization induced by excess metal ions and acid pH. If the negative cooperativity towards metal binding to A sites in doubly metalated forms at pH 8 is in substantial agreement with 113Cd‐NMR data, the equivalence in conformation between Zn2+‐ and Cd2+‐reconstituted alkaline phosphatase emphasizes that no serious structural changes are introduced by the metal replacement.
Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin
Authors: DARDIK Rima , LAHAV Judith
Published: March 2005

Thrombospondin is a large multifunctional glycoprotein synthesized, secreted and incorporated into the extracellular matrix by several cell types in culture. It is also present in the blood platelet and is secreted following platelet activation. We have previously shown that thrombospondin co‐distributes with fibronectin in the extracellular matrix and that it can bind directly to purified fibronectin. In order to elucidate the chemical aspects of thrombospondin incorporation into the extracellular matrix, we studied the interaction of endothelial cell thrombospondin and fibronectin. We find that endothelial cell thrombospondin has two distinct binding domains for fibronectin. One domain is on the 70‐kDa core fragment, probably similar to that of platelet thrombospondin. The other domain is on the 27‐kDa N‐terminal fragment and is unique to endothelial cell thrombospondin. The dissociation constant of the intact endothelial‐cell‐derived molecule is 0.7 ± 0.2 × 10−7 M. Following fragmentation, the separate domains bind with somewhat lower affinity: the core domain binds with a Kd of 3.4 ± 1.5 × 10−7 M and the N‐terminal domain binds with a Kd of 8.8 ± 1.8 × 10−7 M. Binding of the intact molecule is Ca2+‐independent. By contrast, following tryptic fragmentation, binding of the 70‐kDa fragment is practically lost. It can be restored, however, by removal of Ca2+, indicating that the binding site on this domain is either sequestered or becomes so following fragmentation. Heparin, which also binds to both fragments, competed with fibronectin binding to the 27‐kDa fragment but not to the 70‐kDa domain. The fact that heparin also competitively inhibits fibronectin binding of the intact molecule further supports sequestration of the fibronectin‐binding domain on the 70‐kDa core fragment. Our data suggest that endothelial‐cell thrombospondin possesses two distinct binding sites for fibronectin, a low‐affinity constitutively available one and a high‐affinity one, possibly sequestered on the intact unbound molecule.
A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA‐polymerase‐α–primase complex
Authors: HAGEMEIER Annette , GROSSE Frank
Published: March 2005

A ribonuclease H which degrades RNA specifically in RNA‐DNA hybrids and, moreover, stimulates its homologous DNA‐polymerase–primase complex was purified from calf thymus. The enzyme consists of a single polypeptide of molecular mass 78 kDa. It requires divalent cations for activity, and prefers Mg2+ over Mn2+. Ribonuclease H is optimally active at neutral pH and in 75 mM potassium acetate and is strongly sensitive to N‐ethylmaleimide. [3H]Poly(rA) · poly(dT), [3H]poly(rC) · poly(dI), and [3H]RNA · M13‐DNA are degraded to 3–9‐mer oligoribonucleotides with similar kinetics, whereas double‐ or single‐stranded DNA, and double‐ and single‐stranded RNA remain unaffected. The enzyme stimulates in vitro DNA synthesis by the immunoaffinity‐purified calf‐thymus DNA‐polymerase‐α–primase complex threefold. When ribonuclease H is present in a three‐fold molar excess to the polymerase‐primase complex, twice as much primer is formed as in the absence of ribonuclease H. Ribonuclease H also stimulates the elongation rate of DNA polymerase α by a factor of 2–3, independent of whether primase‐primed DNA templates or templates primed with oligonucleotides are used. Our results suggest that this form of ribonuclease H is a likely candidate for a genuine primer‐removing enzyme in mammalian cells.
Structure of Proteus mirabilis 027 O‐specific polysaccharide containing amino acids and phosphoethanolamine
Authors: VINOGRADOV Evgeny V. , KRAJEWSKA‐PIETRASIK Danuta , KACA Wiestaw , SHASHKOV Alexander S. , KNIREL Yuriy A. , KOCHETKOV Nikolay K.
Published: March 2005

The following structure of the repeating unit of the O‐specific polysaccharide of Proteus mirahilis 027, containing amides of d‐glucuronic and n‐galacturonic acids with l‐lysine and l‐alanine, respectively, N‐acetyl‐d‐glucosamine and phosphoethanolamine, has been determined:

image
The main methods used for structural analysis of the polysaccharide were selective solvolysis with anhydrous hydrogen fluoride and Smith degradation, as well as NMR spectroscopy and mass spectrometry.

The immunodominant role of the lateral N‐acetyl‐d‐glucosamine and phosphoethanolamine in manifesting the serological specificity of P. mirubilis 027 has been established.
Neomycin induces high‐affinity agonist binding of G‐protein‐coupled receptors
Authors: HERRMANN Eva , GIERSCHIK Peter , JAKOBS Karl H.
Published: March 2005

Neomycin, an inositol‐phospholipid‐binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G‐protein‐coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1 – 10 mM) was found to induce high‐affinity binding of the chemotactic tripeptide, N‐formyl‐methionylleucylphenylalanine (fMet‐Leu‐Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high‐affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine‐5′‐O‐(3‐thiotriphosphate), to reduce fMet‐Leu‐Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high‐affinity GTPase by fMet‐Leu‐Phe. In close agreement to these findings on the pertussis‐toxin‐sensitive Gi‐protein‐coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high‐affinity agonist binding of Gs‐protein‐coupled β‐adrenoceptors. Similar to formyl peptide receptor binding, high‐affinity binding of isoproterenol to β‐adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine‐5′‐O‐(3‐thiotriphosphate), to a similar maximal extent but with an about 100‐fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high‐affinity agonist binding of Gi‐ and Gs‐protein‐coupled receptors, but not in inducing subsequent G‐protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor‐G‐protein interaction.
Electron transport in sulfate‐reducing bacteria
Authors: STEWART David E. , LEGALL Jean , MOURA Isabel , MOURA José J. G. , PECK Harry D. , XAVIER António V. , WEINER Paul K. , WAMPLER John E.
Published: March 2005

A hypothetical model of the complex formed between the iron‐sulfur protein rubredoxin and the tetraheme cytochrome c3 from the sulfate‐reducing bacteria Desulfovibrio vulgaris (Hildenborough) has been proposed utilizing computer graphic modeling, computational methods and NMR spectroscopy. The proposed complex appears feasible on the basis of complementary electrostatic interaction and steric factors and is consistent with the data from NMR experiments. In this model, the non‐heme iron atom of rubredoxin is in close proximity to heme 1 of cytochrome c3. The complex is stabilized by charge‐pair interactions and hydrogen bonds. This complex is compared to the flavodoxin‐cytochrome c3 complex previously proposed [Stewart, D. E., LeGall, J., Moura, L., Moura, J. J. G., Peck, H. D. Jr, Xavier, A. V., Weiner, P. K. & Wampler, J. E. (1988) Biochemistry 27, 2444–2450] and new NMR data shows that both proteins interact with the same heme group of the cytochrome as postulated.
Engineering of protein bound iron‐sulfur clusters
Authors: BEINERT Helmut , KENNEDY Mary Claire
Published: March 2005

An increasing number of iron‐sulfur (Fe‐S) proteins are found in which the Fe‐S cluster is not involved in net electron transfer, as it is in the majority of Fe‐S proteins. Most of the former are (de)hydratases, of which the most extensively studied is aconitase. Approaches are described and discussed by which the Fe‐S cluster of this enzyme could be brought into states of different structure, ligation, oxidation and isotope composition. The species, so obtained, provided the basis for spectroscopic and chemical investigations. Results from studies by protein chemistry, EPR, Mössbauer, 1H, 2H and 57Fe electron‐nuclear double resonance spectroscopy are described. Conclusions, which bear on the electronic structure of the Fe‐S cluster, enzyme‐substrate interaction and the enzymatic mechanism, were derived from a synopsis of the recent work described here and of previous contributions from several laboratories. These conclusions are discussed and summarized in a final section.
Rapid intramolecular turnover of N‐linked glycans in plasma membrane glycoproteins
Authors: TAUBER Rudolf , PARK Choon‐Sik , BECKER Andreas , GEYER Rudolf , REUTTER Werner
Published: March 2005

Plasma membrane glycoproteins of rat hepatocytes undergo a rapid terminal deglycosylation in that the terminal sugars of the oligosaccharide side chains are rapidly removed from the otherwise intact glycoproteins [Tauber, R., Park, C. S. & Reutter, W. (1983) Proc. Natl Acad. Sci. USA 80, 4026–4029]. The present paper demonstrates that this rapid intramolecular turnover of plasma membrane glycoproteins is not restricted to peripheral sugars but, in contrast to liver, in hepatoma the core sugars of the oligosaccharide chains are also involved. Intramolecular turnover was measured in Morris hepatoma 7777 in five plasma membrane glycoproteins with Mr of 85000 (hgp85), 105000 (hgp105), 115000 (hgp115), 125000 (hgp125), 175000 (hgp175) (hgp = hepatoma glycoprotein) that were isolated and purified to homogeneity by concanavalin‐A—Sepharose affinity chromatography and semipreparative SDS gel electrophoresis. Analysis of the carbohydrates of hgp85, hgp105, hgp115 and hgp125 revealed the presence of N‐linked oligosaccharides containing L‐fucose, D‐galactose, D‐mannose and N‐acetyl‐D‐glucosamine, but only of trace amounts of N‐acetyl‐D‐galactosamine; hgp175 additionally contained significant amounts of N‐acetyl‐D‐galactosamine, indicating the presence of both N‐ and O‐linked oligosaccharides. As shown by digestion with endoglucosaminidase H, the N‐linked oligosaccharides of hgp105, hgp115, hgp125 and hgp175 were of the complex type, whereas hgp85 also contained oligosacharides of the high‐mannose type. Half‐lives of the turnover of the oligosacharide chains and of the protein backbone of the five glycoproteins were measured in the plasma membrane in pulse‐chase experiments in vivo, using L‐[3H]fucose as a marker of terminal sugars, D‐[3H]mannose as marker of a core sugar and L‐[3H]leucine for labelling the protein backbone. Protein backbones of the five glycoproteins were degraded with individual half‐lives ranging over 41–90 h with a mean of 66 h. Compared to the degradation of the polypeptide backbone, both the terminal sugar L‐fucose and the core sugar D‐mannose turned over with much shorter half‐lives averaging about 20 h in the five glycoproteins. The data show that, conversely to liver, within plasma membrane glycoproteins of hepatoma not only peripheral sugars but also core sugars of the oligosaccharides are split off during the life‐span of the protein backbone. It may therefore be assumed that this reprocessing of plasma membrane glycoproteins is sensitive to malignant transformation.
Ceramic hydroxyapatite high‐performance liquid chromatography of complexes membrane protein and sodium dodecylsulfate
Authors: HORIGOME Tsuneyoshi , HIRANUMA Takaaki , SUGANO Hiroshi
Published: March 2005

Ceramic hydroxyapatite high‐performance liquid chromatography was examined as a chromatographic method by which complexes of whole membrane proteins and sodium dodecyl sulfate could be analyzed. The chromatographic conditions were optimized using the erythrocyte membrane as a model. Whole proteins, including membrane proteins larger than 100 kDa, were eluted as sharp peaks from the column and separated well from each other under optimum conditions. This method gave better resolution of protein‐SDS complexes than other chromatographic methods reported so far. The sodium dodecyl sulfate complexes of 24 well characterized proteins were analyzed by this method and their retention times were examined. The positive correlation of the retention time with log (molecular mass) and logΣ (hydrophobicity of amino acids) but not with the isoelectric point, was observed. Based on these results, the mechanism underlying the interaction of protein‐SDS complexes with ceramic hydroxyapatite was discussed.
Conformation of secretin in dimethyl sulfoxide solution
Authors: HOFMANN Mechtild , GONDOL Daniel , BOVERMANN Günter , NILGES Michael
Published: March 2005

The solution conformation of the 27‐residue polypeptide hormone secretin in dimethyl sulfoxide has been determined on the basis of 1H‐NMR measurements. The experimental data set used in the structure determination consisted of 98 nuclear‐Overhauser‐enhancement‐derived interproton and dihedral angle restraints from coupling constants. The NH‐NH and Hα‐NH NOEs were determined from build‐up rates, while the remaining distances were classified in a qualitative manner.

The structure calculations consisted of two phases. First, dynamical simulated annealing calculations were carried out to find conformations of the peptide which satisfy NOE and φ dihedral restraints. The convergence of ten calculated structures was good except for those regions of the molecule where NOE data were not unambiguous. From the calculated set another initial structure was built which was again minimized in several 5‐ps calculations now employing the full empirical energy function. The resulting structures of secretin reveal conformationally well‐defined regions, but not a single uniform secondary structure. The structure is different from the calculated structure from trifluoroethanol/water measurements.
ADP‐ribosylation of the 78‐kDa glucose‐regulated protein during nutritional stress
Authors: LENO Gregory H. , LEDFORD Barry E.
Published: March 2005

Starvation of a mouse hepatoma cell line, Hepa, for any essential amino acid results in the mono‐ADP‐ribosylation of an 80‐kDa protein, P80. The ADP‐ribose acceptor and its putative precursor were identified in two‐dimensional gel patterns and isolated by electroelution. Amino‐terminal sequence analysis showed they were the 78‐kDa glucose‐regulated protein, GRP78. Starvation of Hepa cells for tryptophan or glucose stimulated the relative rate of synthesis, and the ADP‐ribosylation of GRP78. Inhibition of N‐linked glycosylation by treatment with tunicamycin, 2‐deoxy‐d‐glucose or glucosamine stimulated the synthesis of non‐ADP‐ribosylated GRP78 up to sixfold with relatively little effect on its ADP‐ribosylation. Both forms were identified in mouse liver, lung, heart, kidney, spleen and brain.
Sequence analysis of the peptide‐elongation factor EF‐2 gene, downstream from those of ribosomal proteins H‐S12 and H‐S7, from the archaebacterial extreme halophile, Halobacterium halobium
Authors: ITOH Takuzi
Published: March 2005

The gene for the peptide‐elongation factor 2 (EF‐2) was cloned from the archaebacterial extreme halophile Halobacterium halobium and sequenced. The 1013 nucleotides upstream from this gene was two open reading frames similar to ribosomal proteins S12 and S7 from Escherichia coli. Sequence alignment studies showed the halobacterial elongation factor 2 to be equivalent to eukaryotic EF‐2 and eubacterial EF‐G. Sequence similarity to the eukaryotic elongation factor was much higher than to the eubacterial factor. Conserved sequence regions were present within the factor and are likely to constitute functionally important domains. These include the sites of GTP binding and ADP ribosylation by diphtheria toxin.