bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and properties of an iron‐sulfur and FAD‐containing 4‐hydroxybutyryl‐CoA dehydratase/vinylacetyl‐CoA3‐2‐isomerase from Clostridium aminobutyricum
Authors: SCHERF Uwe , BUCKEL Wolfgang 
Published: March 2005

4‐Hydroxybutyryl‐CoA dehydratase, the key enzyme in the metabolism of γ‐aminobutyrate in Clostridium aminobutyricum, represents approximately 15–25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg−1. The dehydratase catalyses the reversible conversion of 4‐hydroxybutyryl‐CoA (Km= 50 μM) to crotonyl‐CoA and possesses a probably intrinsic vinylacetyl‐CoA 3‐2‐isomerase with a specific activity of 223 nkat mg−1. The equilibrium of the reversible dehydration was determined from both sides as K= [crotonyl‐CoA]/[4‐hydroxybutyryl‐CoA] = 4.2±0.3. Cyclopropylcarboxyl‐CoA was not converted to crotonyl‐CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2±0.2 mol FAD (64±5% oxidized). 16±0.8 mol Fe and 14.4±1.2 mol inorganic sulfur, which probably form iron‐sulfur clusters. Exposure to air resulted initially in a slight activation followed by irrevesible inactivation. Concomitantly the vinylacetyl‐CoA ‐isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanofer‐rate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration the dehydration a non activated C‐H bond at C‐3 of 4‐hydroxybutyryl‐CoA has to be cleaved. A putative mechanism for 4‐hydroxybutyryl‐CoA dehydratase is proposed in which this cleavage is achieved by a FAD‐dependent oxidation of 4‐hydroxybutyryl‐CoA to 4‐hydroxycrotonyl‐CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2′ reaction leading to vinylacetyl‐CoA. Finally isomerisation yields crotonyl‐CoA.4‐Hydroxybutyryl‐CoA dehydratase is quite distinct from 3‐hydroxyacyl‐CoA dehydratase (crotonase) and 2‐hydroxyacyl‐CoA dehydratases. Contrary to the latter enzymes [e.g. (R)‐lactyl‐CoA dehydratase and (R)‐2‐hydroxyglutaryl‐CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C‐H bond at C‐3, 4‐hydroxybutyryl‐CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4‐hydroxybutyryl‐xybutyryl‐CoA dehydratase is not inactivated by oxidants such as 5 mM 4‐nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.
Cell Swelling and the sensitivity of autophagic proteolysis to inhibition by amino acids in isolated rat hepatocytes
Authors: MEIJER Alfred J. , GUSTAFSON Lori A. , LUIKEN Joost J. F. P. , BLOMMAART Pietjan J. E. , CARO L. Heleen P. , VAN WOERKOM George M. , SPRONK Claire , BOON Louis 
Published: March 2005

In the isolated perfused rat liver, autophagic proteolysis is inhibited by hypo‐osmotic perfusion media [Häussinger, D., Hallbrucker, C., vom Dahl, S., Lang, F. & Gerok, W. (1990) Biochem. J. 272, 239–242]. Here we report that in isolated hepatocytes, incubated in the absence of amino acids to ensure maximal proteolytic flux, proteolysis was not inhibited by hypo‐osmolarity while the synthesis of glycogen from glucose, a process known to be very sensitive to changes in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955–959], was stimulated under identical conditions. However, in isolated hepatocytes, hypo‐osmolarity increased the sensitivity of autophagic proteolysis to inhibition by low concentrations of amino acids. The anti‐proteolytic effect of hypo‐osmolarity in our experiments was not due to stimulation of amino‐acid transport into the hepatocytes: neither the consumption of most amino acids, nor the rate of urea synthesis was appreciably affected by hypo‐osmotic incubation conditions. In the course of these studies we also found that hypo‐osmolarity increased the affinity of protein synthesis for amino acids.In the presence of amino acids the intracellular level of ATP was not much affected. However, because of cell swelling under these conditions the intracelluar concentration of ATP decreased. It is proposed that a small part of the inhibition of proteolysis by amino acids may be due to this fall in ATP concentration.
Nitric oxide synthase
Authors: RIVEROS‐MORENO Valentina , BEDDELL Christopher , MONCADA Salvador 
Published: March 2005

The amino acid sequence for the constitutive rat brain nitric oxide (NO) synthase was analysed by a set of computer programs that estimate and display physicochemical properties such as hydrophilicity, flexibility, accessibility, hydrophilic periodicity and conformation [Comerford, S. A., McCance, D. J., Dougan, G. & Tite, J. P. (1991) J. Virol. 65, 4681–4690]. Overall, they allow prediction of whether each peptide region will be an α‐helix, a β‐strand or a less regular coil and also whether the region will be buried in the protein core or exposed to water at the surface of the protein molecule. Ten peptide regions were chosen; the majority were predicted to be exposed areas of the molecule and therefore likely to be immunogenic. The peptides were chemically synthesised, coupled to keyhole limpet haemocyanin carrier protein and injected into rabbits to raise antibodies.These antibodies have been used by us and others to locate the NO synthase in different tissues and species. Here we present the characterisation of the antibodies in relation to the possible conformation of the enzyme and an immunological comparison between two isoforms of NO synthase: constitutive (rat brain) and inducible (macrophage). Peptide regions predicted to be exposed, flexible or substantially in core, have produced antibodies that were able to recognise the native protein. Peptides of mixed characteristics possibly involved in the binding site tended to produce antibodies with low recognition for the tertiary structure of the native, purified NO synthase, although these peptides were all highly immunogenic. We postulate that either the peptides when conjugated to the carrier protein attain a different conformation to that in the native NO synthase, or alternatively the accessibility of the antibodies to substrate binding sites is highly restricted by steric hindrance. This latter seems to be more likely since a mixture of antibodies against this area of the protein molecule was able to achieve a similar neutralisation of the enzyme activity as the antibodies against the whole enzyme (∼ 50%).Most of the selected anti‐peptide antibodies were not able to cross‐react with the inducible macrophage enzyme; only two that have 60% sequence identity showed a weak reaction in Western blot. The polyclonal antibody against the complete brain enzyme showed cross‐reaction in a Western blot with inducible enzyme. The macrophage enzyme was able to compete weakly with the binding of the brain enzyme to its own antibody, but 10 times more inducible protein was required. We propose that conformational epitopes are responsible for the cross‐reaction, suggesting that the overall tertiary structures of the two enzymes share a degree of similarity.
Steady‐state kinetics and analysis of pH dependence on wild‐type and a modified allosteric Pseudomonas aeruginosa ornithine carbamoyltransferase containing the replacement of glutamate 105 by alanine
Authors: TRICOT Catherine , NGUYEN Van Thanh , STALON Victor 
Published: March 2005

The substitution of alanine for glutamate at position 105 (E105 A) of the allosteric ornithine carbamoyltransferase (OTCase) of Pseudomonas aeruginosa abolishes the carbamoylphosphate (CP) cooperativity observed in the wild‐type enzyme. A kinetic analysis of [E105 A]OTCase was performed in order to determine the mechanism of the reaction. The results of initial velocity and inhibition studies are consistent with an ordered mechanism with CP as the first substrate to add to the enzyme. In addition, similar studies have been made using the wild‐type enzyme in the presence of the activator, phosphate. The results are similar to those obtained with [E105 A]OTCase indicating that the residue E105 is critical for the allosteric transition of the wild‐type enzyme.The activities of the wild‐type allosteric OTCase and of [E105 A]OTCase have been studied in the pH range 5.8–8.2 in the absence and in the presence of positive and negative effectors. The sigmoid saturation of OTCases by CP has been analyzed according to the Hill equation. At low pH values, CP cooperativity is low in the wild‐type enzyme but cooperativity and [S]CP0.5 values increase markedly with pH. For [E105 A]OTCase, the saturation by CP is hyperbolic at all pH values; in this modified enzyme, the presence of spermidine, an allosteric inhibitor of the wild‐type enzyme, results in an inhibition which induces CP cooperativity. Thus, the ionization of the residue E105 apparently results in a conformational change in the wild‐type enzyme which modifies the catalytic site. Since the [E105 A]enzyme retains the heterotropic effects of the wild‐type enzyme, other structural features are required for the allosteric transition in the wild‐type catabolic OTCase.
Glucose administration induces the premature expression of liver glucokinase gene in newborn rats
Authors: BOSSARD Pascale , PARSA Ramine , DECAUX Jean‐François , IYNEDJIAN Patrick , GIRARD Jean 
Published: March 2005

Glucokinase first appears in the liver of the rat 2 weeks after birth and its activity rapidly increases after weaning on to a high‐carbohydrate diet. The appearance of glucokinase is principally due to the increase of plasma insulin and to the decrease of plasma glucagon concentrations. Oral glucose administration to 1‐ or 10‐day‐old suckling rats induced an increase in plasma insulin and a fall in plasma glucagon and allowed a rapid accumulation of liver glucokinase mRNA, secondarily to a stimulation of gene transcription. When unrestrained late pregnant rats were infused with glucose during 36 h to induce an increase in fetal plasma insulin and a decrease in fetal plasma glucagon concentrations, glucokinase mRNA was detectable in fetal liver but the level was 100‐fold lower than that observed in 1‐ or 10‐day‐old suckling rats. It is suggested that the hormonal environment did not allow glucokinase gene expression to be induced in fetal liver and that the absence of expression of glucokinase in suckling rat liver is due to the presence of low plasma insulin and high plasma glucagon levels.The chromatin structure of the glucokinase gene was examined during development by identification of DNase‐I‐hypersensitive sites from the region comprised between −8 kb upstream and +4 kb downstream of the cap site. Five hypersensitive sites were found: four liver‐specific sites upstream of the cap site and one non‐specific site in the first intron. These sites are already present in term fetus but the intensity of the two proximal sites located upstream of the cap site increase markedly after birth. This suggests that these sites could be implicated in the regulation of glucokinase gene expression by insulin and glucagon. Full DNase‐I‐hypersensitivity of these two proximal sites seems necessary for the mature response of glucokinase gene in response to changes in pancreatic hormones concentrations.
Ferredoxin binding site on ferredoxin: NADP+ reductase
Authors: JELESAROV Ilian , De PASCALIS Antonio R. , KOPPENOL Willem H. , HIRASAWA Masakazu , KNAFF David B. , BOSSHARD Hans Rudolf 
Published: March 2005

The chloroplast enzyme ferredoxin:NADP+ reductase (FNR) catalyzes the reduction of NADP+ by ferredoxin (Fd). FNR and Fd form a 1:1 complex that is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. To localize lysine residues at the Fd binding site of FNR, the FNR:Fd complex (both proteins from spinach) was studied by differential chemical modification. In a first set of experiments, free FNR and the FNR:Fd complex were reacted with the N‐hydroxysuccinimidyl ester of biotin. Biotinylated peptides and non‐biotinylated peptides were separated on monovalent avidin‐Sepharose and purified by high‐performance liquid chromatography. Two peptides containing Lys18 and Lys153, respectively, were less biotinylated in complexed FNR than in free FNR. In a second set of experiments, free and complexed FNR were treated with 4‐N,N‐dimethylaminoazobenzene‐4′‐isothiocyano‐2′‐sulfonic acid (S‐DABITC) to obtain coloured lysine‐modified FNR. Protection of Lys153 was again found by modification with S‐DABITC. In addition, Lys33 and Lys35 were less labelled in the S‐DABITC‐modified, Fd‐bound enzyme. FNR modified in the presence, but not in the absence, of Fd was still able to bind Fd, indicating that the Fd‐protected residues are involved in the formation of the Fd:FNR complex.The lysine residues disclosed by differential modification surround the positive end of the molecular dipole moment (558 Debye ∼ 1.85 × 10−27 Cm) and are located in a domain of strong positive potential on the surface of the FNR molecule. This domain we had proposed to belong to the binding site of FNR for Fd [De Pascalis, A. R., Jelesarov, I., Ackermann, F., Koppenol, W. H., Hirasawa, M., Knaff, D. B. & Bosshard, H. R. (1993) Protein Science 2, 1126–1135]. The prediction was based on the complementarity of shape between positive and negative potential domains of FNR and Fd, respectively.
Recombinant soluble human interleukin‐6 receptor
Authors: STOYAN Tanja , MICHAELIS Uwe , SCHOOLTINK Heidi , VAN DAM Marc , RUDOLPH Rainer , HEINRICH Peter C. , ROSE‐JOHN Stefan 
Published: March 2005

The recombinant soluble human interleukin‐6 receptor (srhIL‐6R) was expressed in Escherichia coli as a non‐glycosylated protein comprising the first 339 amino acids after the signal peptide. The protein accumulated within the cells as insoluble protein aggregates (inclusion bodies). After solubilization, 10% of the denatured srhIL‐6R could be renaturated by an in vitro folding procedure using l‐arginine and the glutathione‐redox system. The native receptors were purified to near homogeneity by affinity chromatography on an IL‐6–Sepharose column.The functional features of the recombinant soluble receptor were further analysed. A part of the extracellular domain (amino acids 145–345) of the human interleukin‐6 receptor (IL‐6R) was expressed in E. coli and the purified protein was used to raise antibodies in rabbits. Characterization of the antiserum obtained indicated that an epitope of 13 amino acids close to the transmembrane region is needed for recognition by the antibodies. Since the antiserum obtained did not interfere with IL‐6 binding, it could be used to establish a cell‐free IL‐6‐binding assay, In this assay, the srhIL‐6R bound IL‐6 with an affinity of Kd= 1.5 nM as measured by Scatchard‐plot analysis. When 125I‐IL‐6 was chemically cross‐linked to the purified srhIL‐6R and analyzed by SDS/PAGE, several 125I‐IL‐6‐containing bands were detected, indicating the possible existence of a multimeric structure of the natural IL‐6/IL‐6R complex. The srhIL‐6R was shown to exhibit biological acitivity, i.e. it stimulated acute‐phase protein synthesis in the recently established human hepatoma cell line HepG2‐IL‐6 which does not express the IL‐6‐binding subunit of the IL‐6R complex on the cell surface.
Isolation and identification of two proto‐oncogene products related to c‐fgr and fyn in a tyrosine‐protein‐kinase fraction of rat spleen
Authors: BRUNATI Anna Maria , JAMES Peter , DONELLA‐DEANA Arianna , MATOSKOVA Brona , ROBBINS Keith C. , PINNA Lorenzo A. 
Published: March 2005

TPK‐III, a tyrosine‐protein‐kinase fraction previously isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451–457] has been further purified and resolved by Mono‐Q FPLC into two homogeneous compounds, Q1 and Q2, with molecular mass approximately 52 kDa, exhibiting high specific activities for phosphate incorporation into the phosphoacceptor substrate poly(Glu80Tyr20) (1194 nmol · min−1· mg−1 and 579 nmol · min−1· mg−1, respectively). Both Q1 and Q2 appear to be scr‐related enzymes since they are readily recognised by anti‐SEEP serum raised against the highly conserved segment at positions 330–345 of p60c‐scr. Q1, but not Q2, interacts with a specific antibody raised against the N‐terminal segment of p55c‐fgr. Microse‐quence analysis of tryptic fragments generated from Q1 revealed five peptides which exactly overlap the expected segments of p55c‐fgr. Two of these peptides were not entirely conserved in any of the other src‐related tyrosine protein kinases. A sixth fragment is very similar, albeit not identical, to the C‐terminus of p55c‐fgr.Microsequence analysis of two tryptic fragments from the other TPK‐III fraction, Q2, provided the sequences FQILNSSE and LTTQETGYIPSNY, which are identical to two segments of fyn not entirely conserved in any of the other src‐related tyrosine protein kinases.These results provide evidence that the spleen tyrosine‐protein‐kinase fraction, conventionally designated TPK‐III, is composed of products from two proto‐oncogenes, one of which corresponds to fyn, while the other is either identical or very closely related to c‐fgr.
The electrochemical‐proton‐gradient‐activated states of F0F1 ATPase in plant mitochondria as revealed by detergents
Authors: VALERIO Marie , DIOLEZ Philippe , HARAUX Francis 
Published: March 2005

ATP hydrolysis, triggered by the addition of polyoxyethylene‐9‐lauryl ether (Lubrol) or lauryldimethylamine oxide (LDAO) to energized plant mitochondria was studied in some details. The membrane disruption was quasi‐instantaneous (2–3 s) with both detergents, as shown by the decrease of turbidity and the stopping of respiration. In pea leaf mitochondria, Lubrol triggered ATP hydrolysis in almost the same way as valinomycin plus nigericin, except that the activity was slightly stimulated and became insensitive to carboxyatractyloside. This allowed investigations of ATP hydrolysis without any interference of the ATP/ADP antiporter or the phosphate carrier. Lubrol did not prevent the ATPase from deactivating in pea leaf mitochondria, and did not trigger any ATP hydrolysis in potato tuber mitochondria. At variance with Lubrol, LDAO changed the properties of the F0F1 ATPase. It made the enzyme oligomycin insensitive and froze it in an activated state. The activity was also 5–8‐times stimulated in pea leaf mitochondria. Moreover, LDAO revealed an important ATP hydrolase activity when added to energized potato tuber mitochondria. Despite the specific effect of LDAO, the activity triggered by this detergent strongly depended on the energized state of the organelles before detergent addition.From this study, it is concluded that the electrochemical proton gradient is completely necessary to activate the F0F1‐ATPase in intact plant mitochondria, as known in chloroplasts and suggested by some reports in animal mitochondria. Moreover, it is suggested that the main difference between the enzymes of pea leaf and potato tuber mitochondria is their rate of deactivation after the collapse of the transmembrane electrochemical potential difference. Finally, when properly used, detergents appear to be a powerful tool to probe the state of the ATPase in intact mitochondria, and maybe in more integrated systems.
Molecular cloning and functional expression of different molecular forms of rat amiloride‐binding proteins
Authors: LINGUEGLIA Eric , RENARD Stephane , VOILLEY Nicolas , WALDMANN Rainer , CHASSANDE Olivier , LAZDUNSKI Michel , BARBRY Pascal 
Published: March 2005

The colon and lung amiloride‐binding proteins were cloned from rat tissues. Two sizes of transcripts were identified. The 2.7‐kb transcript codes for an 85‐kDa protein, whereas the 1.2‐kb transcript codes for a 25‐kDa polypeptide. The 2.7‐kb transcript was detected in the proximal and distal colon and in duodenum, liver, placenta and thymus. The 1.2‐kb transcript was the only form present in lung and spleen, and it was also detected in placenta and colon. The short form corresponds to the 3′ terminus of the longer one. It is formed by alternative transcription under the control of an internal promoter.Cells stably transfected with cDNAs encoding these two proteins were used for binding studies using [3H]phenamil, a potent blocker of the epithelial Na+ channel, derived from amiloride. Both the long and short forms of the protein bind amiloride and some of its derivatives, but they have distinct pharmacologies. The order of potency of the different amiloride derivatives to inhibit [3H]phenamil binding was phenamil (K0.5= 10 nM) > benzamil (K0.5= 43 nM) > amiloride (K0.5= 1.4 μM) ∼ ethylisopropylamiloride (K0.5= 1.6 μM) for the long form, whereas it was phenamil (K0.5= 68 nM) > amiloride (K0.5= 3.2 μM) = ethylisopropylamiloride (K0.5= 4 μM) ∼ benzamil (K0.5= 6.3 μM) for the short form. Although the binding proteins described here are distinct from the pore‐forming protein of the epithelial Na+ channel, the pharmacological profile of the long form of the ABP is identical to that described previously in pig and human kidney, and similar to that expected for an epithelial Na+ channel. The pharmacological profile of the short form resembles that previously described for an amiloride‐binding protein in pneumocytes. Results presented in this paper suggest that previously purified preparations showing Na+ channel activity contain different forms of the amiloride‐binding protein, possibly associated with other proteins.The similarity between amiloride‐binding proteins and a protein identified in seminal vesicles suggests that amiloride‐binding proteins are the first members of a new family of epithelia‐specific proteins.