bluuebunny/crossref_metadata_2025_split huggingface dataset:
The kinetic mechanism of sheep liver sorbitol dehydrogenase
Authors: LINDSTAD Rune I. , HERMANSEN Leonila F. , McKINLEY‐McKEE John S. 
Published: March 2005

The relations between the kinetic parameters for both sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase show that a Theorell‐Chance compulsory order mechanism operates from pH 7.4 to 9.9 This is supported by many parallels with the kinetics of horse liver alcohol dehydrogenase, which operates by this classical mechanism.An isotope‐exchange study using D‐(2H8)sorbitol confirmed the existence of ternary complexes and that, under maximum velocity conditions, their interconversion is not rate‐determining. Substrate inhibition at high concentrations of D‐sorbitol or D‐fructos confirmed rate‐determining enzyme—coenzyme product dissociation, slowed by the existence of more stable abortive ternary enzyme‐conenzyme product complexes with substrate. The effect of the inhibitor/activator 2,2,2‐tribromo‐ethanol showed the existence of enzyme‐NAD‐CBr3CH2OH complexes inhibiting the first phase of reaction and enzyme‐NADH‐CBr3CH2OH complexes dissociating more rapidly than the usual rate‐determining enzyme‐NADH coenzyme product dissociation in the final phase. Inhibition studies with dithiothreitol also confirmed an ordered binding of coenzymes and second substrates to sorbitol dehydrogenase. Neither D‐sorbitol nor D‐fructose had any effect on enzyme inactivation by the affinity labelling reagent DL‐2‐bromo‐3‐(5‐imidazolyl)propionic acid, thus giving no evidence for their existence as binary enzyme‐substrate complexes.Several alternative polyol substrates for sorbitol dehydrogenase gave the same maximum velocity as sorbitol. This indicated a common rate‐limiting binary enzyme‐NADH product dissociation and a similarity of mechanism.An enzyme assay for pH 7.0 and 9.9 is given which enables the concentration of sorbitol dehydrogenase to be determined from initial rate measurements of enzyme activity.
The primary structure of piscine (Oncorhynchus mykiss) retinol‐binding protein and a comparison with the three‐dimensional structure of mammalian retinol‐binding protein
Authors: ZAPPONI Maria Carla , ZANOTTI Giuseppe , STOPPINI Monica , BERNI Rodolfo 
Published: March 2005

The primary structures of two variants of rainbow trout (Oncorhynchus mykiss) plasma retinol‐binding protein (RBP) were determined and found to be approximately 60% identical with those of both human and Xenopus laevis RBPs. The comparable sequence similarities that we have found agree with the estimate of similar divergence times between bony fishes and mammals and between bony fishes and amphibians. The two piscine RBP variants differ by six amino acid substitutions at positions that are not crucial for the interaction with retinol, on the basis of the human RBP three‐dimensional structure [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1990) Proteins Struct. Func. Genet. 8, 44–61].2. Models were developed for the three‐dimensional structures of rainbow trout and X. laevis RBPs, based on that of human RBP. The overall three‐dimensional structure appears to be very well preserved for RBPs isolated from vertebrate species for which the divergence time is 350–400 million years. At variance with an almost absolute conservation for the residues that participate in the formation of the retinol binding site in mammalian RBPs, several amino acid replacements are found for this part of the RBP molecule when the comparison is extended to piscine and amphibian RBPs. However, the only allowed amino acid replacements are either conservative or more than 0.4 nm distant from retinol. Besides the retinol binding site, a few regions at the protein surface appear to be rather conserved during phylogenetic development of vertebrates and, therefore, might be involved in molecular interactions.
The hemoglobins of Notothenia angustata, a temperate fish belonging to a family largely endemic to the Antarctic Ocean
Authors: FAGO Angela , D'AVINO Rossana , DI PRISCO Guido 
Published: March 2005

The blood of the teleost Notothenia angustata contains a major hemoglobin (Hb 1, over 95% of the total), accompanied by a minor component (Hb 2). The two hemoglobins have identical β chains and differ in their α chains. The primary structure of both hemoglobins has been established through the elucidation of the complete amino acid sequence of the three chains. The study of the oxygen‐binding properties shows that Hb 1 displays the Bohr and Root effects and has high affinity for organic phosphates. N. angustata belongs to the family Nototheniidae, suborder Notothenioidei. Unlike the vast majority of nototheniid species, which live in isolation in the Antarctic Ocean and have developed cold adaptation, N. angustata inhabits the waters of southern New Zealand and is not cold adapted. Although some hematological parameters typically favour oxygen transport in a temperate environment, the hemoglobin multiplicity and structural and functional features closely resemble those of the Antarctic species of the same family and suborder. Thus, N. angustata may be considered as a link between temperate and Antarctic habitats. The hypothetical separation history of N. angustata from the Antarctic species of the same family is discussed in the light of the present findings.
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.
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.
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.
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.
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.
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.
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.
Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin‐dependent Na+ pump glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli
Authors: BENDRAT Klaus , BUCKEL Wolfgang 
Published: March 2005

1. The primary sodium‐ion pump glutaconyl‐CoA decarboxylase (GCD) from Acidaminococcus fermentans is composed of four subunits: GCDA, the carboxytransferase (65 kDa), GCDB, the carboxylyase (36 kDa) GCDC, the biotin carrier (24 kDa) and GCDD (14 kDa) of unknown function. A genomic library of A. fermentans was screened with an antiserum raised against whole GCD. A clone giving the strongest reaction in an immunoassay contained a 12‐kbp genomic fragment from A. fermentans and was analysed further. An oligonucleotide deduced from the N‐terminus of GCDA was used for probing the corresponding gene gcdA. It is 1761 bp in length and encodes for a protein of 64.3 kDa. Both partial amino acid sequences obtained from GCDA, the N‐terminus as well as an internal tryptic peptide, were detected in the open reading frame (ORF) of gcdA.2. Sequencing of the flanking regions revealed three adjacent ORF (ORF1–3) which do not code for any of the peptide sequences known of the other GCD subunits. The ORF downstream of gcdA (ORF3) is followed by hgdA and hgdB coding for 2‐hydroxyglutaryl‐CoA dehydratase, the preceeding enzyme of the pathway of glutamate fermentation. Our results suggest that at least these three genes of the hydroxyglutarate pathway are organised in an operon and that the genes of the other GCD subunits from which peptide sequences are known (GCDB and GCDC) are not located adjacent to gcdA.3. gcdA was amplified from genomic DNA using the polymerase chain reaction and cloned into the expression vector pJF118HE. Active GCDA subunit (up to 2.8 nkat/mg protein), catalysing the biotin‐dependent formation of crotonyl‐CoA from glutaconyl‐CoA, was obtained in cell‐free extracts of Escherichia coli DH5α by moderately inducing the tac promoter of pJF118HE with 25–100 μM isopropyl‐1‐thio‐β‐d‐galactoside. Strong induction (1mM isopropyl‐1‐thio‐β‐d‐galactoside) led to the formation of inclusion bodies from which GCDA could not be reactivated. The apparent Km= 51 mM for free biotin of the expressed GCDA subunit with V= 1.9 nkat/mg protein is similar to that of butanol‐treated GCD composed of GCDA and GCDC (apparent Km= 40 mM). Biocytin was found to be a somewhat better carboxy acceptor for the expressed GCDA subunit (apparent Km= 13 mM; V= 1.0 nkat/mg protein).4. Native GCD and expressed GCDA were treated with 2 mM N‐ethylmaleimide showing different kinetics of inactivation: GCD lost half of its activity within 6 min, whereas expressed GCDA required 21 min.