bluuebunny/crossref_metadata_2025 huggingface dataset:
Salt dependence, kinetic properties and catalytic mechanism of N‐formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri
Authors: BREITUNG Jürgen , BÖRNER Gerhard , SCHOLZ Sabine , LINDER Dietmar , STETTER Karl O. , THAUER Rudolf K.
Published: March 2005

N‐Formylmethanofuran(CHO‐MFR): tetrahydromethanopterin(H4MPT) formyltransferase (for‐myltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100‐fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N‐terminal amino acid sequence of the polypeptide was determined.

The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half‐maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revcaled that phosphates and sulfates both affected the Vmax rather than the Km for CHO‐MFR and H4MPT. At the optimal salt concentration and at 65°C, the Vmax was 2700 U/mg (1 U = 1 μmol/min), the Km for CHO‐MFR was 50 μM and the Km for H4MPT was 100 μM. At 90°C, the temperature optimum of the enzyme, the Vmax was about 2.5‐fold higher than at 65°C.

Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90°C decreased in the order K2HPO4= (NH4)2SO4≫ KCI = NH4Cl = NaCl ≫ Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary‐complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.
Peroxisomal acetoacetyl‐CoA thiolase of an n‐alkane‐utilizing yeast, Candida tropicalis
Authors: KURIHARA Tatsuo , UEDA Mitsuyoshi , KANAYAMA Naoki , KONDO Jun , TERANISHI Yutaka , TANAKA Atsuo
Published: March 2005

Two genes encoding acetoacetyl‐CoA thiolase (thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n‐alkane‐utilizing yeast, Candida tropicalis, were isolated from the λEMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N‐terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41 898 Da for thiolase IA and 41 930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl‐CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl‐CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl‐terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, ‐Ala‐Lys‐Leu‐COOH, unlike those of other thiolases reported hitherto.
Purification and characterization of proteinase In, a trypsin‐like proteinase, in Escherichia coli
Authors: KATO Munetoshi , IRISAWA Takaji , OHTANI Makiko , MURAMATU Mutumi
Published: March 2005

We previously found a trypsin‐like proteinase which momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication (Kato, M., Irisawa, T., Morimoto, Y. and Muramatu, M., unpublished results). The proteinase was named proteinase In. It was purified approximately 2880‐fold with a recovery of 15%. The isolated enzyme appeared homogeneous by gel filtration and electrophoresis. Its molecular mass was estimated by analytical gel filtration and SDS/PAGE as approxmately 66 kDa. The isoelectric point of proteinase In is 4.9 and its optimal pH is approximately 9. Although protein In hydrolyzes fluorogenic substrate for trypsin, its hydrolytic activity seems markedly affected by amino‐acid sequence lying towards the N‐terminal from the P1 (lysine, arginine) residue. The proteinase does not hydrolyze N2‐benzoyl‐d,l‐arginine‐4‐nitronanilide and fluorogenic substrates for chymotrypsin and elastase. The proteinase activity is inhibited by leupeptin, antipain and 4‐nitrophenyl 4‐guanidinobenzoate, but the effects of tosyl‐l‐lysine chloromethane, diisopropylfluorophosphate, benzamidine and pentamidine isethionate on the proteinase activity are weak or not inhibitory. Its activity is strongly affected in the presence of NaCl and KCl, and at a concentration of 1.5 M, these increase the activity 14‐fold and 13‐fold, respectively, above that without salt. Proteinase In was strongly inhibited by various esters of trans‐4‐guanidinomethylcyclohexanecarboxylic acid, and their inhibitory effects were roughly correlated with those on growth of E. coli. Proteinase activity was found in the cytoplasmic fraction.
A myofibrillar protein phosphatase from rabbit skeletal muscle contains the β isoform of protein phosphatase‐1 complexed to a regulatory subunit which greatly enhances the dephosphorylation of myosin
Authors: DENT Paul , MACDOUGALL Lindsay K. , MACKINTOSH Carol , CAMPBELL David G. , COHEN Philip
Published: March 2005

A form of protein phosphatase‐1 (PP1M), which possesses 25‐fold higher activity towards the P light chain of myosin (in heavy meromyosin) than other forms of protein phosphatase‐1, was purified over 200 000‐fold from the myofibrillar fraction of rabbit skeletal muscle. PP1M, which eluted from Superose 12 with an apparent molecular mass of 60 kDa, was dissociated by LiBr into two subunits. One of these displayed enzymic properties identical to those of the catalytic subunit of protein phosphatase‐1 (PP1C) and was identified as the β isoform of PP1C by amino acid sequencing. The second subunit had no intrinsic protein phosphatase activity, but greatly increased the rate at which PP1C dephosphorylated skeletal‐muscle heavy meromyosin and decreased the rate at which it dephosphorylated glycogen phosphorylase.

The properties of PP1M, together with those of smooth muscle PP1M [Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. (1992) Eur. J. Biochem. 210, 1023–1035] and the previously characterised glycogen‐associated form of protein phosphatase‐1 (PP1G), indicate that the subcellular localisation and substrate specificity of PP1 is determined by its interaction with specific targetting subunits.
Identification and isolation of common and tissue‐specific geranylgeranylated γ subunits of guanine‐nucleotide‐binding regulatory proteins in various tissues
Authors: MORISHITA Rika , FUKADA Yoshitaka , KOKAME Koichi , YOSHIZAWA Tôru , MASUDA Katsuyoshi , NIWA Masatake , KATO Kanefusa , ASANO Tomiko
Published: March 2005

Heterotrimeric guanine‐nucleotide‐binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their α subunits, though a notable multiplicity of γ subunits has also been demonstrated. To investigate whether each subtype of α subunit is associated with a particular γ subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than tow kinds of γ subunit. Of the brain G proteins, GoA, GoB, and Gil contain the same set of three γ subunits, but Gi2 contains only two of these subunits. Lung Gil and Gi2 and spleen Gi2 and Gi3 had similar sets of two γ subunits, one of which was distinct from the γ subunits of brain G proteins. These observations indicate indicate that each subtype of α subunit is associated with a variety of βγ subunits, and that the combinations differ among cells. For analyses of the structural diversity of the γ subunits, βγ subunits were purified from the total G proteins of each tissue and subjected to reverse‐phase HPLC under denaturing conditions, where none of the β subunits were eluted from the column. Three distincy γ subunits were isolated in this way from brain βγ subunits. In contrast, lung and spleen βγ subunits contained at least five γ subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several γ subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant γ subunit in each tissue was identical, and the sequences coincided exactly with that of ‘γ6’ [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758–15761]. Fast‐atom‐bombardment mass spectrometry analysis indicated that this abundant γ subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C‐terminus, as was ‘γ6’ from brain. In addition to abundant γ subunits, other tissue‐specific γ subunits were also shown to be geranylgeranylated by gas‐chromatographycoupled mass spectrometry analysis of Raney nickel‐treated γ subunits. These results suggest that most γ subunits associated with many different subtypes of α subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated γ subunit.
Protein interaction with ice
Authors: HEW Choy L. , YANG Daniel S. C.
Published: March 2005

Many organisms have evolved novel mechanisms to minimize freezing injury due to extracellular ice formation. This article reviews our present knowledge on the structure and mode of action of two types of proteins capable of ice interaction. The antifreeze proteins inhibit ice crystal formation and alter ice growth habits. The ice nucleation proteins, on the other hand, provide a proper template to stimulate ice growth. The potential applications of these proteins in different industries are discussed.
Mitogen‐activated‐protein‐kinase‐catalyzed phosphorylation of microtubule‐associated proteins, microtubule‐associated protein 2 and microtubule‐associated protein 4, induces an alteration in their function
Authors: HOSHI Minako , OHTA Kunihiro , GOTOH Yukiko , MORI Akiko , MUROFUSHI Hiromu , SAKAI Hikoichi , NISHIDA Eisuke
Published: March 2005

Mitogen‐activated protein kinase (MAPK), a serine/threonine‐specific protein kinase which is generally activated by stimulation with various growth factors and phorbol esters, utilizes microtubule‐associated protein (MAP) 2 as a good substrate in vitro. We have found that MAPK‐catalyzed phosphorylation of MAP2 resulted in a significant loss in its ability to induce tubulin polymerization. The chymotryptic fragments, containing a microtubule‐binding domain of MAP2, were phosphorylated by MAPK and the ability of the fragments to induce tubulin polymerization was also greatly decreased by the phosphorylation, suggesting that phosphorylation of the microtubulebinding domain is important for functional alteration of MAP2. In addition to MAP2, a 190‐kDa heat‐stable MAP (MAP4) found in various tissues and cells, was a good substrate for MAPK in vitro. Phosphorylation of MAP4 inactivated tubulin polymerization. We examined the effect of phosphorylation of MAP2 and MAP4 on the dynamics of microtubules nucleated by purified centrosomes in vitro. The data showed that MAPK‐catalyzed phosphorylation of MAP2 and MAP4 reduced their ability to increase the apparent elongation rate and the number of microtubules nucleated by the centrosome. Thus, MAPK is capable of phosphorylating MAPs and negatively regulating their microtubule‐stabilizing function.
Localization of the intrachain disulfide bonds of the envelope glycoprotein 71 from Friend murine leukemia virus
Authors: LINDER Monica , LINDER Dietmar , HAHNEN Josef , SCHOTT Hans‐Henning , STIRM Stephan
Published: March 2005

Envelope glycoprotein 71 from Friend murine leukemia virus was purified to homogeneity by reversed‐phase HPLC. It could be shown that all 20 cysteine residues of the molecule are linked by disulfide bonds. After complete tryptic digestion, peptide containing cystine were identified by comparison of the reversed‐phase HPLC profile of the digest with that of a reduced aliquot which had been subjected to affinity chromatography on thiol‐Sepharose. The locations of the 10 disulfide bonds were determined by isolation, further digestion and analysis of peptides containing cystine.

The first cysteine residue of the sequence (Cys46) was shown to be coupled to the sixth (Cys98), leading to a large loop containing four additional cysteine residues. Computer model building and energy calculations led to the assignment of Cys72 to Cys87 and Cys73 to Cys83. The following four cysteine residues of the sequence also constitute a structural unit, with Cys121 bonded to Cys141 and Cys133 to Cys146, and the last two cysteine residues in the amino‐terminal domain of glycoprotein 71 form a small loop (Cys178 to Cys184). The first two cysteine residues of the carboxy‐terminal domain produce a very small hydrophobic loop (Cys312–Cys315). Cys361 is bound to Cys373, Cys342 to Cys396 and Cys403 to Cys416. A model for the folding pattern of the viral glycoprotein is proposed.
Unresolved rearrangement of thiazoline form of glutathione
Authors: FUJII Katsuhiko
Published: March 2005

The thiazoline form of glutathione was investigated with regard to its unresolved instability under neutral conditions. A simple method was developed for production of the thiazoline in stable solid form, thereby facilitating preparation of its neutral solution without using excess base and enabling isolation of the reaction product in quantity by ion‐exchange chromatography. Analysis of the product by HPLC, infrared and ultraviolet absorption spectroscopy, mass spectrometry and proton magnetic resonance led to the identification of a cyclic amidine form of glutathione. The instability of the thiazoline is, therefore, due to an intramolecular rearrangement reaction, rather than hydrolysis. Once formed, the amidine is stable at pH 5–7 and in concentrated HCl, showing no tendency to rearrange back to either the original thiazoline or glutathione under these conditions.
Changes of the human liver GM3 ganglioside molecular species during aging
Authors: RIBONI Laura , ACQUOTTI Domenico , CASELLATO Riccardo , GHIDONI Riccardo , MONTAGNOLO Gianguido , BENEVENTO Angelo , ZECCA Luigi , RUBINO Federico , SONNINO Sandro
Published: March 2005

Sialosyl‐lactosylceramide, GM3, is the major ganglioside of human liver, where it constitutes more than 90% of the total lipid‐bound sialic acid. When analyzed by thin‐layer chromatography, human liver GM3 migrates as two main spots. They are representative of ganglioside molecular species which differ in the acyl moiety. The faster running spot is mainly composed of molecular species with non‐hydroxylated C22–C24 acyl chains; the other contains mainly molecular species bearing nonhydroxylated C16–C18 and α‐hydroxylated C16–C24 acyl chains.

In this study the content of the two GM3 molecular species groups was investigated in 31 subjects ranging from 19 to 85 years of age. By thin‐layer chromatography we observed that the group of molecular species containing non‐hydroxylated C22–C24 acyl chains, decreased linearly with subject age, while that of non‐hydroxylated C16–C18 acyl chains and hydroxylated C16–C24 acyl chains increased linearly.

Fast‐atom‐bombardment mass spectrometry performed on seven samples from subjects ranging from 21 to 78 years of age demonstrated that the age‐dependent increase of the lower spot is caused by an increase in the hydroxylated fatty acid form of GM3, the content of non‐hydroxylated C16–C18 fatty acid species remaining constant with age.
Structural investigation on the lipopolysaccharide of Escherichia coli rough mutant F653 representing the R3 core type
Authors: HAISHIMA Yuji , HOLST Otto , BRADE Helmut
Published: March 2005

The chemical structure of the core oligosaccharide of the lipopolysaccharide isolated from Escherichia coli rough mutant F653, representing the enterobacterial R3 core type, was investigated by quantitative and methylation analyses, nuclear magnetic resonance spectroscopy, gas‐liquid chromatography/mass spectrometry, and determined as

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All sugars are present as α‐pyranosides but the anomeric configurations of the 3‐deoxy‐D‐manno‐octulopyranosonic acid (Kdo) residues could not be determined. The third Kdo and the heptose‐linked GlcN residue are present in nonstoichiometric amounts; the GlcN residues may be, at least partially, N‐acetylated.
X‐ray diffraction study of the interaction between carboxypeptidase A and (S)‐(+)‐1‐amino‐2‐phenylethyl phosphonic acid
Authors: MANGANI Stefano , CARLONI Paolo , ORIOLI Pierluigi
Published: March 2005

The structure of the carboxypeptidase A complex with the inhibitor (S)‐(+)‐1‐amino‐2‐phenylethylphosphonic acid has been determined at 0.23 nm resolution. The ΔF map shows electrondensity peaks both in the S1 and S'1 sites, where the inhibitor molecule can be modeled in two different orientations with approximate 50% occupancy. In the proposed model, the phosphonate group binds to the zinc in a monodentate fashion. Other anchoring groups for the inhibitor molecule are Arg127 (hydrogen bonds with the phosphonate oxygen atoms) and Glu270 (hydrogen bond with the amino group in one of the two orientations).

A recent spectroscopic investigation of the complex between cobalt(II) carboxypeptidase A and (S)‐(+)‐1‐amino‐2‐phenylethylphosphonic acid is essentially in agreement with our results.
Characterization of heterotrimeric collagen molecules in a sea‐pen (Cnidaria, Octocorallia)
Authors: TILLET‐BARRET Emmanuelle , FRANC Jean‐Marie , FRANC Suzanne , GARRONE Robert
Published: March 2005

The collagen of a primitive invertebrate, the sea‐pen Veretillum Cnidaria, Octocorallia), was studied with respect to its molecular‐chain composition. The soft extracellular tissues (mesoglea) were solubilized by limited pepsin proteolysis and the collagen was isolated by selective precipitaion at 0.7 M NaCl under acidic conditions.

The pepsinized molecules were 260 nm in length, as demonstrated by electron microscope studies of rotary‐shadowed molecules and of the segment‐long‐spacing crystallites obtained by dialysis against ATP.

SDS/PAGE of the extract produced two main bands susceptible to bacterial collagenase, designated as the α1 and α2 chain, which were differentiated clearly by their CNBr cleavage products and the higher glycosylation rate of the α2 chain. The latter finding corresponds with the high hydroxylysine content of the α2 chain. The α1/α2 chain ratio observed in SDS/PAGE and the fact that only one peak was obtained by concanavalin‐A affinity chromatography of a non‐denatured 0.7 M NaCl extract demonstrate the α1 [α2]2 molecular structure of this collagen.

These results contrast with data on the structure of other coelenterates (i.e.[α]3 for sea anemone collagen molecules and α1α2α3 for jellyfish collagen molecules). They are discussed in relation to the evolution of collagen.
Investigation of the mechanisms of irreversible thermoinactivation of Bacillus stearothermophilusα‐amylase
Authors: BROSNAN Marian P. , KELLY Catherine T. , FOGARTY William M.
Published: March 2005

Bacillus stearothermophilus NCIB 11412 produces a highly thermostable α‐amylase. The enzyme displayed half‐lives of irreversible thermoinactivation at 90°C of 1.9 min and 12.5 min at pH 5.0 and pH 8.0, respectively. Molecular mechanisms of irreversible thermoinactivation were investigated. At both pH 5.0 and pH 8.0 irreversible thermoinactivation was due to heat‐induced breakdown of non‐covalent interaction within the protein molecule, resulting in unfolding and consequent formation of altered structures. Hydrophobic interactions were shown to be the most important non‐covalent mechanisms involved in this phenomenon. Although not dramatically effecting the rates of irreversible thermoinactivation, electrostatic interactions, including hydrogen bonding, were also shown to have a contributary role in this process. At pH 8.0 a covalent mechanism, that of oxidation of thiols was also shown to be of secondary importance to hydrophobic interactions in the irreversible thermoinactivation of this enzyme.
Determinants on simian virus 40 large T antigen are important for recognition and phosphorylation by casein kinase I
Authors: UMPHRESS Jason L. , TUAZON Polygena T. , CHEN Charng‐Jui , TRAUGH Jolinda A.
Published: March 2005

Casein kinase I has been shown to phosphorylate Ser123 and possibly Thr124, in simian virus 40 (SV40) large T antigen; the same sites are also modified in cultured cells incubated with 32Pi [Friedrich A. Grässer, Karl H. Scheidtmann, Polygena T. Tuazon, Jolinda A. Traugh & Gernot Walter (1988) Virology 165, 13–22]. The peptide, A‐D‐S‐Q‐H‐S‐T‐P‐P, which corresponds to the amino acid sequence 118–125 of SV40 large T antigen, was synthesized together with peptides containing changes in specific amino acid residues on either side of Ser123. These peptides were used as model substrates to determine the amino acids in the SV40 large T antigen important for recognition by casein kinase I. The native peptide identified above, with aspartate at the −4 position, was a poor substrate for casein kinase I in vitro. Peptides with acidic residues added at the −2 and −3 positions, preceding Ser123, were phosphorylated by casein kinase I with apparent Km values around 2 mM and Vmax values up to 500 pmol · min−1· ml−1. When acidic residues were added at both sides of the phosphorylatable serine, the peptide had a first‐order rate constant over 20‐fold higher than peptides with acidic amino acid residues at the N‐terminus only; the apparent Km value was 0.65 mM with a Vmax of 2900 pmol · min−1· ml−1. The effects of modifying Ser120 to phosphoserine were examined by addition of a recognition sequence for the cAMP‐dependent protein kinase prior to Ser120. Prior phosphorylation of the peptide at Ser120 lowered the apparent Km to 0.061 mM and increased the Vmax to 360 pmol · min−1· ml−1, a 50‐fold decrease in Km for casein kinase I and a 6‐fold increase in Vmax as compared to the non‐phosphorylated peptide. This indicates that Ser120, which has been shown to be phosphorylated in vivo, provides an appropriate recognition determinant for casein kinase I.
Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775
Authors: SNOEP Jacky L. , WESTPHAL Adrie H. , BENEN Jacques A. E. , TEIXEIRA de MATTOS M. Joost , NEIJSSEL Oense M. , DE KOK Arie
Published: March 2005

In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram‐positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1α and E1β), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram‐positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions.
Neutral oligosaccharides of bovine submaxillary mucin
Authors: CHAI Wengang , HOUNSELL Elizabeth F. , CASHMORE Geoffrey C. , ROSANKIEWICZ Jerzy R. , BAUER Christopher J. , FEENEY James , FEIZI Ten , LAWSON Alexander M.
Published: March 2005

Twenty‐two neutral O‐linked oligosaccharides ranging from monosaccharides to octasaccharids were identified in bovine submaxillary‐gland‐mucin glycoprotein by a combination of liquid secondary‐ion mass spectrometery, methylation analysis and 1H‐NMR. Only five of these have been previously detected in bovine submaxillary‐gland mucin although several have been described from other sources of mucin. The structures include short linear sequences 3‐linked to N‐acetylgalactosaminitol (GalNAcol) and branched structures based on either a GlcNAc(β1–6)[Gal(β1–3)]GalNAcol or GlcNAc(β1–6)[GlcNAc(β1–3)]GalNAcol core region. Oligosaccharides not previously characterised from any source were the disaccharide GalNAcα1–6GalNAcol (GalNAc, N‐acetylgalactosamine and the hexasaccharide GlcNAc(β1–6){GalNAc(α1–3)[Fuc(α1–2)]Gal(β1–4)GlcNAc(β1–3)}GalNAcol (Fuc, l‐fucose). Oligosaccharides of the blood‐group‐A type have not been detected previously in bovine submaxillary‐gland mucin although their occurrence on bovine gastric‐mucosal glycoproteins has been established by classical immunochemical studies.
N‐linked glycopeptides with blood group determinants lacking neuraminic acid from the epithelial cells of rat small and large intestine
Authors: NORRSELL Henrik , BENGTSSON Jonas , JOVALL Per‐Åke , HANSSON Gunnar C.
Published: March 2005

The N‐linked type of glycans were prepared as their glycopeptides after pronase digestion of the epithelial cells from the small and large intestine of two inbred strains of rat. These glycopeptides were analysed for sugar composition, for blood‐group activity, by 1H‐NMR spectroscopy, and after permethylation by electron‐impact mass spectrometry. The glycopeptides were of the triantennary and tetraantennary types with intersected GlcNAc. The terminal parts were, in contrast to most N‐linked glycans, devoid of neuraminic acid residues. Instead they contained blood‐group determinants. Blood‐group‐H types 1 (Fucα1 – 2Galβ1 – 3GlcNAc) and 2 (Fucα1 – 2Galβ1 – 4GlcNAc) were found in the small and large intestines of both strains, although type‐1 predominated. One rat strain (GOT‐W) did not express blood‐group‐A glycopeptides in the small intestine, but the large intestine from the same strain did. The other strain (GOT‐BW) expressed blood‐group‐A determinants in the small intestine. The lack of neuraminic acid residues in the small and large intestine and of blood‐group‐B activity in the large intestine differed from that found in glycosphingolipids obtained from the same organs.
Baculovirus‐mediated expression and characterisation of rat glycogen synthase kinase‐3β, the mammalian homologue of the Drosophila melanogaster zeste‐white 3sgg, homeotic gene product
Authors: HUGHES Kenneth , PULVERER Bernd J. , THEOCHAROUS Panteli , WOODGETT James R.
Published: March 2005

Molecular cloning of glycogen synthase kinase‐3 (GSK‐3) has demonstrated the existence of a novel form, termed GSK‐3β, which is highly related to the well characterised GSK‐3α protein but derived from a distinct gene. The cDNA cloning also revealed a striking degree of amino acid identity between the two GSK‐3 proteins, particularly the β‐form, and the zeste‐white3/shaggy (zw3sgg) homeotic gene of Drosophila melanogaster. Abrogation of zw3sgg causes pleiotropic effects on fruitfly development affecting segmental organisation and cell fate determination. In view of the potential importance of GSK‐3β in mammalian development and the lack of previous characterisation, we have expressed this protein in insect cells using recombinant baculovirus. A rapid purification scheme has been developed yielding essentially pure GSK‐3β protein in three chromatographic steps. The protein has autonomous protein kinase activity and similar, but not identical, substrate preferences to GSK‐3α. Both GSK‐3 proteins activate the MgATP‐dependent form of protein phosphatase‐1 and thus display ‘factor A’ activity. Since GSK‐3β exhibits an identical site specificity to GSK‐3α with respect to phosphorylation of the proto‐oncogene/transcription factors c‐jun and c‐myc, it is likely that the Drosophila zw3sgg protein kinase has a similar specificity for such transcription factors which may underlie the pleiotropic phenotypes observed when the Drosophila homologue is mutationally inactivated.
Advances in metallo‐procarboxypeptidases
Authors: AVILÉS Francesc X. , VENDRELL Josep , GUASCH Alicia , COLL Miquel , HUBER Robert
Published: March 2005

Our knowledge on the structure and functionality of pancreatic carboxypeptidases is rapidly expanding to include that of their zymogen forms. The recent application of fast and mild isolation procedures, together with modern molecular genetic and biochemical‐biophysical characterization approaches, has provided a clearer view of the basic structures and functional states in which these zymogens occur, and their evolutionary relationships. The same holds for related metallocarboxypeptidases, either in the pro or active forms, that have been isolated and characterized in non‐digestive fluids and tissues, where they probably play an important role in protein and peptide processing. The determination of the three‐dimensional structure of the A and B pancreatic zymogens has revealed the molecular determinants of their inactivity and proteolytic activation. The folding of their 95‐residue activation segment in a globular N‐terminal domain (74–81 residues) and in a connecting region (20–14 residues), and the specific contacts of these pieces with the substrate binding sites of the enzyme, are important factors in zymogen inhibition. On the other hand, the different length of the α‐helical connecting region and the stability of its contacts with the enzyme account for the different activation properties of A and B zymogens.
DNA excision‐repair synthesis is enhanced in a murine leukemia L1210 cell line resistant to cisplatin
Authors: CALSOU Patrick , BARRET Jean‐Marc , CROS Suzanne , SALLES Bernard
Published: March 2005

Among various molecular mechanisms of cel resistance to antitumor agents such as cisplatin, it has recently been suggested that enhanced DNA‐repair activity might be involved in the resistant phenotype of cell lines. Mouse leukemia‐cisplatin‐resistant cell lines L1210/10 (adapted in vitro) and L1210/DDP (adapted in vivo) have been reported to exhibit an increase DNA‐ repair activity, as determined by host‐cell reactivation after transformation with damaged plasmids. In this paper, excision‐repair activity was monitored by an in‐votro assay allowing quantification of DNA‐repair synthesis in cell extracts from resistant and sensitive parental cells (L1210/10 versus L1210/0 and L1210/DDP versus L1210/S). Experimental conditions for optimal repair‐synthesis activity were found to be different from these reported with human cell‐line extracts. L1210/S sensitive cell line, grown in vivo by a weekly intraperitoneal graft in mice, sdisplayed a repair activity about fourfold lower than the same cell line maintained in vitro or than L1210/0 cell grown in votro. The repair activity was found similar in a L1210/10 and L1210/0 cell lines, but it was enhanced in L1210/DDP resistant cell line when compared with its parental line.
Osteogenesis imprefecta and type‐I collagen mutations
Authors: VALLI Maurizia , SANGALLI Antonella , ROSSI Antonio , MOTTES Monica , FORLINO Antonella , TENNI Ruggero , PIGNATTI Pier Franco , CETTA Giuseppe
Published: March 2005

In this study we describe a new dominant point mutation in COL1A1 causing a lethal form of Osteogenesis imperfecta (type II B). Dermal cultured fibroblasts from the proband were shown to produce both normal and hevily overmodified type‐I collagen. The mutation introduced a local conformational perturbation, which causes abonormal exposure of arginine residues; the triple helical domain was susceptible to trypsin digestion even at 30°C. The chains bearing the point mutationwere poorly secreted and short‐term pulse experiments showed that the extensive intracellular retention of mutant trimers also imparied the secretion of mormal chains.

The molecular defect was localized in a COL1A1 allele by cloning and sequencing a cDNA region corresponding to the CB6 peptide. A G to C transversion which causes the substitution in the triple helical region of Gly910 with alanine was found. The mutation also causes the disappearance of a MspI‐recognition site at nucleotide 3263 of the proα1(I) coding sequence. Restriction analysis, along with the biochemical screening of collagens, allowed us to perform prenatal diagnosis on cells from chorionic‐villus sampling and to exclude the recurrence of the mutation in the sibling.
The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes
Authors: MIGNOGNA Giuseppina , ALLOCATI Nerino , ACETO Antonio , PICCOLOMINI Raffaele , DI ILIO Carmine , BARRA Donatella , MARTINI Filippo
Published: March 2005

The complete amino acid sequence of glutathione transferase from Proteus mirabilis was determined. The sequence was reconstructed by analysis of peptides obtides obtained after cleavabge by trypsin, Glu‐C and Asp‐N endoproteinases. The enzyme subunit is composed of 203 amino acid residues corresponding to a molecular mass of 22856 Da. Comparison of this sequence with other known primary structures of the corresponding enzyme from different sources shows a low level of identity (17–26%) with only seven conserved residues in all the sequences considered. This novel glutathione transferase could represent the prototype of a new class, possibly including other bacterial enzymes.
Changes in the insulin‐sensitive glycosyl‐phosphatidyl‐inositol signalling system with aging in rat hepatocytes
Authors: SÁNCHEZ‐ARIAS Juan A. , SÁNCHEZ‐GUTIÉRREZ Julio C. , GUADAÑO Ana , ALVAREZ José F. , SAMPER Begoña , MATO José M. , FELÍU Juan E.
Published: March 2005

An inositol‐phosphate glycan (InsP glycan), which is the polar head group of an insulin‐sensitive glycosyl‐phosphatidylinositol (glycosyl‐PtdIns), has been reported to mimic some insulin actions when added to different types of cells. In connection with this, a specific, time‐dependent and energy‐dependent transport system for this InsP glycan has been identified in isolated rat hepatocytes [Alvarez, J. F., Sánchez‐Arias, J. A., Guadaño, A., Estevez, F., Varela, I., Felíu, J. E. & Mato, J. M. (1991) Biochem. J. 274, 369–374]. Here we have investigated the glycosyl‐PtdIns‐dependent insulin‐signalling system in hepatocytes isolated from either 3‐month‐old or 24‐month‐old rats. Aging reduced the stimulatory effect of insulin on [U‐14C]glucose incorporation into glycogen, caused a significant decrease in basal glycosyl‐PtdIns levels and blocked the insulin‐mediated hydrolysis of this lipid. In 24‐month‐old rats, we also observed a diminution in the rate of hepatocyte InsP‐glycan uptake and a marked reduction of the stimulatory effect of this compound on glycogen synthesis. These results support the hypothesis that insulin resistance associated with aging is accompanied by an impairment of the glycosyl‐PtdIns‐dependent cellular signalling system.
Mutant aspartate aminotransferase (K258H) without pyridoxal‐5′‐phosphate‐binding lysine residue
Authors: ZIAK Martin , JÄGER Joachim , MALASHKEVICH Vladimir N. , GEHRING Heinz , JAUSSI Rolf , JANSONIUS Johan N. , CHRISTEN Philipp
Published: March 2005

If the pyridoxal‐phosphate‐binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P. (1990) Eur. J. Biochem. 187, 329–333]. The three‐dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild‐type enzymes, small conformational differences being restricted to parts of the active site. If aspartate or glutamate was added to the pyridoxal from of the mutant enzyme [λmax 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (λmax= 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2‐oxoglutarate to the pyridoxamine form (λmax 330 nm, positive CD), a putative ketimine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild‐type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25°C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with [4′‐3H]pyridoxamine 5′‐phosphate resulted in rapid release of 3H with a first‐order rate constant k′= 5X10−4s−1 similar to that of the wild‐type enzyme.

Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the activesite lysine residue. The imidazole ring of H258, however, seems too distant from Cα and C4′ to act efficiently as proton donor/acceptor in the aldimine–ketimine tautomerization, suggesting that the prototiopic shift might be mediated by an intervening water molecule. Transimination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.
Localization and functional analysis of DNase‐I‐hypersensitive sites in the human c‐sis/PDGF‐B gene transcription unit and its flanking regions
Authors: DIRKS Ron P. H. , JANSEN Hans J. , GERRITSMA Jort , ONNEKINK Carla , BLOEMERS Henri P. J.
Published: March 2005

We studied the regulation of the expression of the human c‐sis/PDGF‐B gene in the following panel of cell lines: K562 cells, in which expression is inducible by phorbol esters; cytotrophoblastderived cell lines JEG‐3 and JAR; carcinoma‐derived cell lines PC3, T24 and HeLa, which show extensive differences in c‐sis mRNA content; dermal fibroblasts, which do not express the gene. We demonstrate that the wide variety of levels of c‐sis mRNA in these cells in mainly determined at the transcription level. Extensive gene rearrangements or amplifications, or significant differences in the stability of the c‐sis transcript could not be found. In fibroblasts and placenta cell lines, inaccessibility of the c‐sis promoter, rather than the absence of transcription factors that activate it, inhibits expression of the endogenous gene. Examination of the chromatin structure of the transcription unit and immediate flanking regions revealed several cell‐type‐specific DNase‐I‐hypersensitivity (DH) sites. Functional analysis of genomic fragments harbouring one or more DH sites showed the presence of negative regulatory elements within intron 1, and of an activating element downstream of the gene. A DH site, located immediately downstream of the promoter in dermal fibroblasts, may regulate accessibility of the promoter by means of specific nucleosome phasing.
Use of gene fusions of the structural gene sdaA to purify L‐serine deaminase 1 from Escherichia coli K‐12
Authors: SU Hongsheng , MONIAKIS John , NEWMAN E. B.
Published: March 2005

The purification by affinity chromatography of β‐galactosidase from strains carrying sdaA/lacZ gene fusions results in the copurification of L‐serine deaminase 1. We conclude that sdaA is the structural gene for the latter enzyme. The purified L‐serine deaminase 1 obtained after collagenase treatment of an sdaA‐collagen‐lacZ fusion differs from the native enzyme by the addition of several amino acids at the C‐terminal. Like the enzyme in crude extracts, this purified enzyme is catalytically inactive, and is activated by incubation with iron and dithiothreitol.
Purification and characterisation of an initiation‐factor‐2 kinase from uninduced mouse erythroleukaemia cells
Authors: MELLOR Harry , PRICE Nigel T. , OLDFIELD Susan , SARRE Thomas F. , PROUD Christopher G.
Published: March 2005

Mouse erythroleukaemia (MEL) cells, which have not been induced into erythroid development, contain a protein kinase (MKu) which phosphorylates the α subunit of protein‐synthesis‐initiation factor 2 (eIF‐2α). In this paper, we show that this kinase phosphorylates both eIF‐2α and a synthetic peptide based on the phosphorylation site in eIF‐2α at Ser51, the target residue for other eIF‐2α kinases. Consistent with this, prior treatment of eIF‐2 with MKu impaired the exchange of bound GDP for GTP which is catalysed by the exchange factor eIF‐2B.

Using a modified cell‐free translation system, we have shown that MKu inhibits translation, consistent with the above observations concerning the site of phosphorylation and the effect of phosphorylation on eIF‐2B‐mediated guanine‐nucleotide exchange.

MKu has been purified and its properties have been compared with those of the haem‐controlled repressor eIF‐2α kinase (HCR) from rabbit reticulocytes. Its behaviour on gel filtration is similar to that of HCR, while its behaviour on anion exchange resembles that of certain phosphorylated species of HCR. Highly purified preparations of MKu contain a protein with an apparent molecular mass of 98 kDa which comigrates with HCR on SDS/PAGE. This protein undergoes phosphorylation when incubated in the presence of Mg2+‐ATP, and both this apparent autophosphorylation and the activity of the kinase against eIF‐2α are inhibited by the same, low, (10 μM) concentrations of haemin. Phosphorylation of the 98‐kDa components present in the MEL‐cell kinase preparation and in purified rabbit reticulocyte HCR occurs on serine and threonine residues. Analysis of these phosphoproteins by peptide mapping reveals significant differences in their structures, indicating that they may be closely related, but are certainly not identical.
Isolation and molecular characterization of a hyperglycemic neuropeptide from the sinus gland of the terrestrial isopod Armadillidium vulgare (Crustacea)
Authors: MARTIN Gilbert , SOROKINE Odile , VAN DORSSELAER Alain
Published: March 2005

The major peptide from the sinus gland of the terrestrial isopod Armadillidium vulgare (Crustacea) has been extracted and purified by reverse‐phase HPLC. This neuropeptide exhibited a high hyperglycemic activity and was therefore named A. vulgare crustacean hyperglycemic hormone (Arv‐CHH). Its average molecular mass measured by mass spectrometry was 8729.3 Da. Its complete amino acid sequence was determined by a combination of Edman degradation and mass spectrometry. The N‐terminal amino acid was found to be unblocked, the C‐terminal residue was found amidated and none of the other 72 residues was affected by any post‐translational modification. Disulfide bond assignment was made unambigously by mass spectromety and Edman degradation was performed on peptides produced by enzymatic cleavage. Relationships with other, similar neuropeptides from decapod sinus glands are discussed.
Purification, characterization, crystallisation and X‐ray analysis of selenomethionine‐labelled hydroxymethylbilane synthase from Escherichia coli
Authors: HÄDENER Alfons , MATZINGER Peter K. , MALASHKEVICH Vladimir N. , LOUIE Gordon V. , WOOD Stephen P. , OLIVER Philip , ALEFOUNDER Peter R. , PITT Andrew R. , ABELL Chris , BATTERSBY Alan R.
Published: March 2005

Hydroxymethylbilane synthase (HMBS) catalyses the conversion of porphobilinogen into hydroxymethylbilane, a linear tetrapyrrolic intermediate in the biosynthesis of haems, chlorophylls, vitamin B12 and related macrocyles. In the course of an investigation of the crystal structure of this enzyme, we intended to follow a new strategy to obtain the X‐ray phase information, i. e. the collection of multiwavelength anomalous diffraction data from a crystal of a seleno‐l‐methionine (SeMet)‐labelled variant of the protein. We have expressed and purified HMBS from Escherichia coli (34268 Da) in which all (six) methionine (Met) residues are replaced by SeMet. Complete replacement, as shown by amino acid composition analysis and by electrospray mass spectrometry, was achieved by growing the Met‐requiring mutant E. coli PO1562 carrying the plasmid pPA410 in a medium containing 50 mg/l SeMet as the sole source of Met. [SeMet]HMBS exhibits full enzyme activity, as reflected by unchanged steady‐state kinetic parameters relative to native enzyme. Rhombohedral crystals of [SeMet]HMBS could be grown at the pH optimum (7.4) of the enzyme (solutions containing 30 mg/ml protein, 0.4 mM EDTA, 20 mM dithiothreitol, 3 M NaCl and 15 mM Bistris‐propane buffer were equilibrated by vapour diffusion at 20°C against reservours of saturated NaCl); However being very thin plates, these crystals were not suitable for X‐ray analysis. Alternatively, rectangular craystals were obtained at pH 5.3 using conditions based on those reported for wile‐type HMBS [sitting drops of 50 μl containing 6–7 mg/ml protein, 0.3, < EDTA, 15 mM dithiothreitol, 10% (mass/vol.) poly(ethylene glycol) 6000 and 0.01% NaN3 in 0.1 M sodium acetate were equilibrated by vapour diffusion at 20°C aginst a reservoir of 10–20 mg solid dithiothreitol]. X‐ray difracion data of the crystals were complete to 93.8% at 0.21 nm resolution and showed that [SeMet]HMBS crystallise isomorphously. A difference Fourier map using FSeMet–Fnative and phases derived from the native structure, which has recently been determined independently by multiple isomorphous replacemetn, showed positive difference peaks centered at or closeto where the sulphur atmos of the Met side chains apper in the native structure. In addition, paird positive/negative peaks in the difference map near the cofactor of HMBS indicate conformational difference in the active site, probable dir to diffrences in the state of oxidaion of the cofactor in two crystalline samples.