bluuebunny/crossref_metadata_2025_split huggingface dataset:
The pH in reversed micelles as imposed by the dihydrogen/proton redox cuple and indicated by viologens and cytochrome c3 using hydrogenase as redox catalys
Authors: van DIJK Cees , SPRUIJT Ruud , LAANE Colja , VEEGER Cees 
Published: March 2005

The pH values in reversed micelles were measured, making use of the hydrogenase enzyme as redox catalyst short‐circuiting the viologen oxidized/semiquinone redox states. The hydrogenases from Desulfovibrio vulgaris (Hildenborough) and from Megasphaera elsdenii were applled. The observed pH values in reversed micelles were not dependent onthe type of hydrogenase.Two cationic [cetyltrimethylammonium bromide and dodecylammonium propionate (DAP)] and two anionic sodiumdodecyl sulphate, sodium di(ethylhexyl)sufosuccinate types of reversed micelles were used in combination with viologens having distiguishable valencies. It was observed that, in the cationic‐reversed micelles, the dissociation constant for the semiquinone dimer had about the same value as compared to bulk water, while this value was significantly higher in the anionic‐reversed micelles. Furthemore, the dissociation Constant was independent of the concentration of viologen semiquinone in the reversed micell, indicating that exchange kinetics are faster than the dimerisation process. With the exception of DAP, a linear relation exists, pH = a · pHrm+ b, between the pH of the bulk water and the pH as measured in the reversed micelle(pHrm). In all these case the value of a is smaller than unity, the value of b ranges between 1.6–2.7. For DAP the pHrm is always around 7. In DAP‐reversed micelles, the counter‐ion propionate probably serves as an internal buffer. Using cytochrome c3, as pH indicator in combination with N,N′‐di(3‐aminopropl)‐4,4′‐bipyridinium)4 + to take care of electron transfer, in cetyltrimethylammonium‐bromide‐reversed micelles the pHrm is about the same as indicated by the viologen; in SDS‐reversed micelles the pHrm is always lower than that incidated by N,N′‐di(3‐aminopropyl)‐4,4′‐pyridinium4 +. In contrast to cytochrome c3 from D. vulgaris, which in reversed micelles cannot become reduced directly by its D. Vularis hydrogenase, the hydrogenase of M. elsdenii is able to reduce its ferredoxin directly.
A mutation at Gly314 of the β subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)‐induced conformational change
Authors: AHMAD Suhail , GLAVAS Natalie A. , BRAGG Philip D. 
Published: March 2005

Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity. A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the β subunit by a Glu residue. The mutant enzyme was partially purified and its trypsin cleavage products examined. The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin. However, the β subunit of the mutant enzyme retained its ability to bind to NAD‐agarose. Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site‐directed mutagenesis. All substitutions for Gly314 abolished the activity completely. The enzyme containing the Gly314→Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314→Glu mutation. It is concluded that the mutation in the β subunit abolished the NADP(H)‐induced conformational change in the mutant enzyme. This conformational change, caused by NADP(H) binding, is required to cleave the normal β subunit at Arg265 by trypsin.The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence [Clarke et al. (1986) Eur. J. Biochem. 158, 647–653].
Purification and characterization of a recombinant murine interleukin‐6
Authors: ZHANG Jian‐guo , MORITZ Robert L. , REID Gavin E. , WARD Larry D. , SIMPSON Richard J. 
Published: March 2005

Murine interleukin‐6 (IL‐6), when expressed in Escherichia coli using the pUC9 vector, accumulated as insoluble aggregates or ‘inclusion bodies’. After selective urea washing of the inclusion bodies, to remove extraneous proteins, murine IL‐6 was solubilized with 8 M guanidine hydrochloride and then rapidly purified to homogeneity by gel‐permeation chromatography followed by reversed‐phase HPLC. It was demonstrated that complete disulfide bond formation in murine IL‐6 occurred during the early urea washing/guanidine hydrochloride extraction steps, so no refolding step was required. When fully reduced murine IL‐6 was dissolved in 8 M guanidine hydrochloride and allowed to airoxidize, complete disulfide bond formation, monitored by analytical reversed‐phase HPLC, was shown to occur within 13 h at 6°C.About 25 mg pure protein was obtained from 37 g wet cells. This recombinant murine IL‐6 had a specific activity in the hybridoma growth factor assay of 2×108 U/mg, which is equivalent to that of native murine IL‐6.During the purification procedure, a number of variant forms of murine IL‐6 were isolated and partially characterized. Two of these forms, T1 and T3, were C‐terminal deletants of murine IL‐6 lacking about 60 and 20 amino acids from the C‐terminus, respectively, while the other form, T2, was an N‐terminal deletant lacking 37 amino acids from the N‐terminus. None of these variant forms of murine IL‐6 bound to the murine IL‐6 receptor and, consequently, all were inactive in the hybridoma growth factor assay.
Biosynthesis of vitamin B12
Authors: LINGENS Brigitte , SCHILD Thomas A. , VOGLER Bernhard , RENZ Paul 
Published: March 2005

The 5,6‐dimethylbenzimidazole moiety of vitamin B12 is formed from riboflavin in aerobic and some aerotolerant bacteria. Thereby C1′ of riboflavin is transformed into C2 of the vitamin B12 base. In the present publication a study on this transformation with riboflavin 2H‐labeled in the 1′R or 1′S position is described. This study was undertaken in order to find out if one of the two hydrogens at C1′ is transferred to C2 of 5,6‐dimethylbenzimidazole.The 2H‐labeled riboflavin samples were synthesized starting from unlabeled or 1‐2H‐labeled ribose and 3,4‐dimethylaniline yielding N‐β‐d‐ribopyranosyl‐3,4‐dimethylaniline. The unlabeled riboside was reduced to N‐d‐ribityl‐3,4‐dimethylaniline with sodium cyanoborotrideuteride, the 2H‐labeled riboside with sodium cyanoborohydride. The ribityl derivatives were transformed into N‐d‐ribityl‐2‐phenylazo‐4,5‐dimethylaniline, and condensed with barbituric acid to riboflavin.The reduction of the ribosyl compound to the ribityl derivtive is only partially stereospecific. Thus the riboflavin synthesized from unlabeled ribose had a 2H ratio of 3/1 (1′R/1′S), the riboflavin obtained from d‐[1‐2H1]ribose of 1/3 (1′R/1′S). The 2H content in these positions was determined from the 1H‐NMR spectra. These spectra showed also that 1 mol 2H/mol riboflavin was present in position 1′.The deuterated riboflavin samples were incubated under aerobic conditions with broken cell preparations of Propionibacterium shermanii. The deuterium content of the 5,6‐dimethylbenzimidazole isolated was determined by mass spectrometry and by 1H NMR. These measurements revealed that the hydrogen in the pro‐S position at C1′ of riboflavin is retained during 5,6‐dimethylbenzimidazole formation, and is thus found at C2 of this base.
Inhibition of calcium‐dependent release of noradrenaline from PC12 cells by botulinum type‐A neurotoxin
Authors: SHONE Clifford C. , MELLING Jack 
Published: March 2005

(a) Clostridium botulinum type‐A neurotoxin (BoNTA) inhibited the calcium‐dependent release of noradrenaline from PC12 cells in a dose‐dependent manner. Under conditions in which intact PC12 cells were incubated with BoNTA for 20 h at 37°C, a neurotoxin concentration of approximately 0.12 ± 0.03 μM was required to inhibit 50% of the calcium‐dependent noradrenaline release.(b) PC12 cells, differentiated in the presence of nerve growth factor for 14 days, showed a similar dose‐dependent inhibition of noradrenaline release by BoNTA with unchanged sensitivity. No specific saturable binding of 125I‐labelled BoNTA was observed to either differentiated or undifferentiated PC12 cells, suggesting a lack of high‐affinity acceptors on the cell surface for the neurotoxin. It is proposed that BoNTA enters PC12 cells either by non‐specific binding to the cell membrane or via a low‐concentration low‐affinity acceptor molecule.(c) A study of the long‐term effects of BoNTA on noradrenaline release from PC12 cells showed that the neurotoxin remains active within the growing cells for several days. Noradrenaline release from PC12 cells exposed to BoNTA (0 3 μM) for 24 h was reduced to less than 20% of control values over a subsequent 4‐day period. After 8 days, release levels were significantly lower (60–65%) than control values, despite a more than 10‐fold increase in the cell mass.(d) Investigation of the subcellular distribution of BoNTA after incubation with PC12 cells for 96 h revealed the bulk of the toxin (94–98%) to be associated with the cell membrane fraction. Of this, 50–80% of the BoNTA was associated with the nuclear and cell debris fraction and 11–25% was recovered in the large‐granule‐vesicle fraction; the specific binding of the neurotoxin to these membrane fractions was found to be similar.(e) Examination of the form of the cell‐associated BoNTA after incubation for 96 h with PC12 cells revealed no evidence of any significant degradation of either neurotoxin subunit. This suggests that the neurotoxin adopts a relatively stable form within the cell. On SDS/PAGE under non‐reducing conditions, no trace of protein bands corresponding to either of the BoNTA subunits were observed, suggesting that little or none of the neurotoxin subunits exists in a monomeric form within the cells. A considerable portion (50–70%) of the membrane‐associated BoNTA was found to be present in the form of large disulphide‐linked aggregates which, on SDS/PAGE in the presence of a thiol, dissociated into the bands corresponding to the neurotoxin heavy‐chain and light‐chain subunits. Examination of the neurotoxin aggregates associated with the dense‐core‐vesicle fraction showed them to contain a greater proportion of the light‐chain subunit (29.3%± 10.3) compared to the control toxin.(f) The above observations are discussed in relation to the mode of action of BoNTA.
A strong protein‐tyrosine kinase activity is associated with a baculovirus‐expressed chicken tkl gene
Authors: GÄRTNER Thomas , KÜHNEL Herbert , RAAB Gerhard , RAAB Monika , STREBHARDT Klaus , RÜBSAMEN‐WAIGMANN Helga 
Published: March 2005

We have previously described a gene named tkl (tyrosine kinase related to lck). It belongs to the src family of protein‐tyrosine kinases and among these it has significant homology to the lck gene (lymphoide cell kinase). The tkl gene product may represent the avian homolog of Lck, which is believed to participate in a lymphocyte‐specific signal transduction pathway by association with a membrane receptor.To study the biochemical properties of the protein, a nearly complete tkl gene (isolated from a cDNA library from chicken spleen cells) was expressed in a baculovirus system. Approximately 10% of the extracted protein consisted of the soluble 51‐kDa Tkl protein (p51tkl) at 40 h post‐infection. This protein was found to be phosphorylated on tyrosine and serine residues at a ratio of 5:1. As expected, glycosylation or myristoylation could not be detected. Immunocomplex kinase assays indicated strong autophosphorylation of p51tkl at tyrosine residues and phosphorylation of exogenous substrates such as D‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), histones H2b and H4, and casein. This protein‐tyrosine kinase activity also exhibited a marked preference for Mn2+ compared to Mg2+. The high level expression of enzymatically active Tkl should provide an excellent tool to further study the biological functions of this class of enzymes.
A study of the hinge‐bending mechanism of yeast 3‐phosphoglycerate kinase
Authors: DRYDEN David T. F. , VARLEY Paul G. , PAIN Roger H. 
Published: March 2005

The hinge‐bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site‐directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C‐terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding.The effect of substrates on the conformation of PGK was studied using 8‐anilino‐1‐naphtha‐lenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C‐terminal domain, and 8‐[2‐[(iodoacetyl)ethyl]amino]naphthalene (I‐AEDANS), attached to Cys197 on the N‐terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1–5° change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS.The enzyme activity of PGK shows a break in the Arrhenius plot at 20°C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine.The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.
Preferential binding of insulin‐like growth factor‐II (IGF‐II) to a putative α2β2 IGF‐II receptor type in C2 myoblasts
Authors: DOMEYNE Anne , PINSET Christian , MONTARRAS Didier , GARANDEL Véronique , ROSENFELD Ron G. , BARENTON Bruno 
Published: March 2005

We have studied insulin‐like‐growth‐factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 μM) or lower concentrations of IGF‐I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF‐II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5).Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF‐I (Kd 0.8–1.2 nM). Affinity cross‐linking of [125I]IGF‐I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the α subunit of the IGF‐I receptor.In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF‐II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF‐II/M6P receptor. This was confirmed by affinity cross‐linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non‐reducing conditions, respectively.In contrast, competitive inhibition of [125I]IGF‐II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF‐II/M6P receptor (Kd 0.2 nM) as evidenced by cross‐linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF‐I than by IGF‐II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non‐reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF‐II/M6P‐receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF‐II binding to this atypical receptor.It emerges from these experiments that C2 cells express a putative α2β2 IGF‐II receptor structurally related to the insulin/IGF‐I receptor family. It is present in myoblasts but not in myotubes. The possible relationship between expression of this receptor and expression of MyoD1 as well as myoblast differentiation is discussed.
Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl‐CoA hydrolase
Authors: LILL Ute , LEFRANK Sigrid , HENSCHEN Agnes , EGGERER Hermann 
Published: March 2005

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl‐CoA + oxaloacetate + H2O → citrate + CoASH) to 4% but did not affect the hydrolysis of citryl‐CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well.2. Tryptic hydrolysis occurs at the N‐terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues.3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue‐Sepharose and differentiated by their isolelectric points (pI= 6.68 ± 0.08 and pI= 6.37 ± 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N‐terminus which is blocked in the native enzyme only.4. Edman degradation of the large tryptic fragment yielded the N‐terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N‐terminal region (positions 9–32) of citrate synthase from Thermoplasma acidophilum.5. The conversion of citrate synthase into essentially a citryl‐CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N‐terminal small peptide.
Characterization of the cytochrome P‐450IID subfamily in bovine liver
Authors: TSUNEOKA Yutaka , MATSUO Yoshinori , HIGUCHI Ryota , ICHIKAWA Yoshiyuki 
Published: March 2005

To elucidate the molecular mechanisms underlying drug detoxification, the structures of the members of the microsomal cytochrome P‐450IID subfamily were analyzed by isolating, mapping and sequencing cytochrome P‐450IID (CYP2D) cDNA clones from bovine liver. The screening was performed under nonstringent conditions so that most of the P‐450IID subfamily members could be obtained. 114 of the 147 positive clones were classified into four groups on the basis of their restriction‐enzyme maps. The maps of the four groups were highly similar, however, the clones of one group contained an insertion of approximately 500 bp in the coding region. Analysis of partial nucleotide sequences of several representative clones from each group showed that the bovine P‐450IID subfamily in liver consisted of several, not many, highly similar members, differing by less than 7% in their nucleotide sequences.The location of the insertion found in the minor group corresponded to intron 7 and the GT/AG rule was found at the exon/intron boundary, suggesting that intron 7 was retained in this group.The complete nucleotide sequences of two clones from the major group were examined to determine the structures of the P‐450IID subfamily in bovine liver. A full‐length cDNA clone (1615 bp) and a partial cDNA clone (1538 bp) contained open reading frames encoding 500 and 487 amino acid residues, respectively. The partial clone lacked the nucleotide sequence corresponding to the first 13 N‐terminal amino acid residues. The deduced amino acid sequences of the two clones were 98% similar, and 80% and 68% similar to those from human CYP2D6 and rat CYP2D1, respectively. Comparisons of the amino acid sequences of the P‐450IID subfamily members showed the highly conserved C‐terminal region of their molecules and the high similarity between the members in one species, especially in cattle and man.
Altered plasma membrane H+‐ATPase from the Dio‐9‐resistant pmal‐2 mutant of Schizosaccharomyces pombe
Authors: GHISLAIN Michel , DE SADELEER Myriam , GOFFEAU André 
Published: March 2005

The pmal‐2 mutation affecting the plasma membrane H+‐ATPase of Schizosaccharomyces pombe has been selected for resistance to the antibiotic Dio‐9. In membrane fractions purified from glucose‐starved cells, the mutant ATPase activity is reduced by 96%, is insensitive to inhibition by vanadate and has a pH profile displaced in the acidic pH range when compared to the wild type. The maximum velocity of the H+‐ATPase activity of plasma membranes from glucose‐activated pmal‐2 cells is activated 20‐fold. This is in striking contrast with the wild‐type ATPase activity, the maximal velocity of which is not affected by glucose. However, similar to the wild‐type enzyme, glucose activation of the pmal‐2 mutant H+‐ATPase reduces the Km for MgATP 9–2 mM and shifts the optimal pH from 4.8 to 6.0–6.5.The pmal‐2 mutation modifies Lys250 to a threonine, which is highly conserved in fungal and plant H+‐ATPases. These results, compared to those reported for mutations of neighbour residues in yeast or mammalian P‐type ATPases, suggest that Lys250 could play a significant role, not only in phosphate binding and/or in the E1P‐E2P conformational isomerisation, but also in glucose activation of the H+‐ATPase.