bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational changes in proteins induced by dynamic associations
Authors: GABELLIERI Edi , STRAMBINI Giovanni B. 
Published: March 2005

Random collisions between macromolecules lead to dynamic associations (lengthy encounters) that in principle could affect their conformation and, in the case of enzymes, their binding and catalytic properties. Exploiting the unique sensitivity of the phosphorescence lifetime, τ, of Trp to the internal flexibility of globular proteins we probed the perturbations induced in the structure of the coenzyme‐binding domain of alcohol dehydrogenase (LADH) and glyceraldehyde‐3‐phosphate dehydrogenase (GraPDH) by the presence in solution of other dehydrogenases and of functionally unrelated proteins. With Trp314 of LADH, the results emphasize that while τ is not affected by the concentration of LADH itself, the addition of micromolar quantities of other proteins causes a distinct reduction in it. From the linear increase of 1/τ with protein concentration one obtains values for the apparent second‐order Stern‐Volmer rate constant that range between 2–200 × 103 M−1 s−1, decreasing 2–3‐fold when ternary complexes of LADH with NADH or NAD+ and inhibitors are involved. Similar effects were observed with Trp310 of GraPDH except that with sorbitol dehydrogenase as perturbant the increase of 1/τ is hyperbolic and governed by an apparent dissociation constant of about 1 μM. Finally, glycerol‐3‐phosphate dehydrogenase, the strongest perturber of both LADH and GraPDH, has either no effect on lactic dehydrogenase from pig heart or induces a moderate lengthening of the triplet lifetime of the rabbit muscle enzyme.Because Stern‐Volmer behaviour is typical also of diffusion‐mediated quenching reactions, a parallel investigation with cysteine, cystine and N‐acetyl‐tryptophanamide demonstrated that among potential, protein‐associated, quenching moieties namely, ‐SH, ‐S‐S‐ and indole groups, only the latter has rate constants approaching the magnitude of protein perturbants. Since considerable evidence rules out the predominance of such quenching reactions, these findings confirm a subtle form of communication between protein molecules in solution. The lack of specificity and the similar effects between dehydrogenases with right and wrong stereospecificity for direct coenzyme transfer suggests that the perturbations monitored are unrelated to this function.
Purification and characterization of a protein‐tyrosine kinase p72syk from porcine spleen
Authors: YANG Cheng , YANAGI Shigeru , WANG Xiaoying , SAKAI Keiko , TANIGUCHI Takanobu , YAMAMURA Hirohei 
Published: March 2005

We have succeeded in purifying p72syk, a non‐receptor‐type protein‐tyrosine kinase carrying high susceptibility to proteolysis [Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S. and Yamamura, H. (1991) J. Biol. Chem. 266, 15790–15796] from porcine spleen. The purification procedure involves a sequential column chromatography, following extraction with 0.5 M NaCl from spleen homogenate, on phosphocellose, Sephacryl S‐200, heparin‐Sepharose CL‐6B, Mono Q and Mono S. SDS/PAGE of the final purified sample revealed a 72‐kDa protein band with about 95% purity and immunodepletion analysis showed immunological cross‐reactivity with anti‐p72syk antibody which does not recognize ZAP‐70. It was purified approximately 3000‐fold with an overall yield of 0.54% according to [Val5]angiotensin II phosphorylation activity and the specific activity of the final sample (30 nmol phosphate min–1) was relatively lower than that of the 40‐kDa kinase, a catalytic fragment of p72syk which lacks two src homology regions 2 domains.The p72syk had an autophosphorylation activity that was performed by intramolecular catalysis accompanied by a phosphate exchange reaction, and could efficiently phosphorylate tubulin, myelin basic protein and H2B histone. Employing [Val5]angiotensin II as a substrate, the apparent Km value for the peptide was 0.91 mM and that for ATP was 0.48 μM. Mn2+, Mg2+ and Co2+ were effective divalent cations and optimum pH was around 8.0–8.5 for the expression of the activity.These results suggest that the purified p72syk may exist as a less active form compared with the 40‐kDa kinase and that the part ofp72syk containing two src homology region 2 domains may participate in the regulation of its activity though the enzymic character is quite similar to that of the 40‐kDa kinase.
Diversity in the processing events at the N‐terminus of type‐V collagen
Authors: MORADI‐AMÉLI Mahnaz , ROUSSEAU Jean‐Charles , KLEMAN Jean‐Philippe , CHAMPLIAUD Marie‐France , BOUTILLON Marguerite‐Marie , BERNILLON Jacques , WALLACH Jean , VAN DER REST Michel 
Published: March 2005

The processing of human collagen type‐V chains was studied using anti‐peptide polyclonal antibodies raised against peptide sequences at the N‐terminal non‐triple‐helical region of pro‐α1(V) and pro‐α2(V) chains. The anti‐peptide polyclonal antibody raised against positions 48–57 of the N‐terminal α2(V) sequence recognized the mature from of the human α2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro‐α2(V) and pN‐α2(V) collagen chains secreted in the cell‐culture media of the rhabdomyosarcoma A204 cell line. The pN‐α2(V) collagen chain from this cell line migrated during electrophoresis with the α2(V) chain obtained from tissues. This demonstrates that the α2(V) chain in tissues is incompletely processed and is present as the pN‐α2(V) collagen chain which lacks the C‐propeptide. In comparison, an anti‐peptide polyclonal antibody raised against residues at positions 284–299 of the N‐terminal α1(V) human sequence failed to recognize the mature form of the α1(V) chain while it reacted with the pN‐α1(V) collagen chain form. These results suggest that the α1(V) chain undergoes a processing event in the N‐terminal region that involves the revmoval of at least the first 284 residues.Amino acid sequence analysis was performed on cyanogen‐bromide‐generated or trypsin‐generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower‐migrating band corresponding to the intact α1(V) chain gave, as expected, only sequences corresponding to the α1(V) chain. However, the band previously considered to be the intact α2(V) chain also gave sequences for the α1(V) chain in addition to the α2(V) chain. This result indicates the presence in tissue extracts of a further processed form of α1(V) chain which migrates with the intact α2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non‐collagenous regions, two bands were observed with an α1(V)/α2(V) chain ratio of 3:1. These results indicate that the α1(V) chain exists in an additional stoichiometry, different from [α1(V)]2α2(V). We suggest the existence of two different populations of type‐V collagen molecules consisting of an [α1(V)]2α2(V) heterotrimer bearing considerable N‐terminal non‐triple‐helical extensions of both α1(V) and α2(V) chains and an [α1(V)]3 homotrimer composed of fully processed α1(V) chains.
Analysis of cytochrome‐b amino acid residues forming the contact face with the iron‐sulfur subunit of ubiquinol:cytochrome‐c reductase in Saccharomyces cerevisiae
Authors: GIESSLER Astrid , GEIER Birgitta M. , de RAGO Jean‐Paul , SLONIMSKI Piotr P. , VON JAGOW Gebhard 
Published: March 2005

Four mutations in the mitochondrial cytochrome b of Saccharomyces cerevisiae have been characterized with respect to catalytic properties, inhibitor resistance and subunit interaction. The respiratory‐deficient mutant [G137E]cytochrome b and the pseudo‐wild‐type revertant [G137E, N256K]cytochrome b were described previously [di Rago, J.‐P., Netter, P. & Slonimski, P. P. (1990) J. Biol. Chem. 265, 3332–3339; di Rago, J.‐P., Netter, P. & Slonimski, P. P. (1990) J. Biol. Chem. 265, 15750–15757]. Two new mutants [N256K]cytochrome b and [N256I]cytochrome b were isolated by dissociation of the second‐site suppressor from the original target mutation.The mutants [G137E]cytochrome b and [G137E, N256K]cytochrome b exhibited a high resistance against methoxyacrylate inhibitors, whereas the suppressors [N256K]cytochrome b and [N256I]cytochrome b showed only a slight resistance. Remarkably, all mutants exhibited stigmatellin cross‐resistance.The electron‐transfer activity from the substrate nonylubiquinol to cytochrome c of mitochondrial membranes was diminished in all mutants. The substitution G137→E decreases Vmax/Km by one order of magnitude, indicating a reduced catalytic efficiency for ubiquinol. The amino acid exchange at position 256 to a positively charged lysine residue or to a hydrophobic isoleucine residue resulted mainly in a diminished specific activity.The iron‐sulfur subunit and the 8.5‐kDa subunit were detectable in all mutants at normal levels in immunoblots of membrane preparations, indicating proper assembly of the complex. However, after purification, the mutant bc1 complex lacked the iron‐sulfur subunit and the 8.5‐kDa subunit. In contrast, the iron‐sulfur subunit can only be dissociated from the parental bc1 complex by harsh treatment.These data suggest that residues 137 and 256 in cytochrome b are crucial for cytochrome‐b/iron‐sulfur protein interaction.
Identification of two antibody‐interaction sites on the surface of Panulirus interruptus hemocyanin
Authors: de HAAS Felix , PERTON Frank G. , van BREEMEN Jan F. L. , DIJKEMA Jan H. , BEINTEMA Jaap J. , van BRUGGEN Ernst F. J. 
Published: March 2005

Negatively stained complexes of Panulirus interruptus (spiny lobster) hemocyanin with two different monoclonal antibodies, named E and J, were studied by electron microscopy and image processing. The attachment site of the antibodies to the hexameric hemocyanin molecule was deduced from two perpendicular views of hemocyanin/antibody complexes, in which either the threefold axis or one of the twofold axes was oriented perpendicular to the supporting film. Images of complexes in these orientations were searched with reference images simulated from the known X‐ray structure of P. interruptus hemocyanin.The two sites were further characterized by combining our results from electron microscopy with structural data obtained by X‐ray diffraction and other methods. These two antibodies recognize different non‐overlapping epitopes. The epitope for clone E is located on domain 3 at the surface of the β barrel and consists of certain loops, which form connections between β‐strand structures. The epitope for clone J is situated on domain 1 at the surface of an α‐helical region and consists mainly of certain α‐helices connecting loops.The orientation of the hemocyanin hexamers in the two complexes is very different, as is demonstrated most clearly when they form chains. Clone E forms complexes with the threefold axes perpendicular to the chain direction, while for clone J the threefold axes seem to be parallel to the main direction. The angle between the Fab part of an IgG molecule and the threefold axis of the hexamer is 60±5° for clone E and 35±7° for clone J. This observation is clearly related to the difference in orientation of the hexamers for the two complexes.
Preparation and structural analysis of oligosaccharide monophosphates obtained from the lipopolysaccharide of recombinant strains of Salmonella minnesota and Escherichia coli expressing the genus‐specific epitope of Chlamydia lipopolysaccharide
Authors: HOLST Otto , THOMAS‐OATES Jane E. , BRADE Helmut 
Published: March 2005

The lipopolysaccharide of the recombinant strain Salmonella minnesota r595–207 expressing the genus‐specific epitope of Chlamydia lipopolysaccharide [Holst, O., Brade, L., Kosma, P. and Brade, H. (1991) J. Bacteriol. 173, 1862–1866] was sequentially de‐O‐ and de‐N‐acylated by mild hydrazinolysis and treatment with 4 M KOH, respectively. The resulting mixture of compounds was separated by high‐performance anion‐exchange chromatography and gel‐permeation chromatography, yielding four oligosaccharide phosphates two of which were readily identified by their 1H‐NMR‐ and 13C‐NMR spectra as α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α‐D‐GlcpN 1,4′‐bisphosphate (tetrasaccharide bisphosphate; Kdo = 3‐deoxy‐D‐manno‐octulopyranosonic acid) and α‐Kdo‐(2–8)‐α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α‐D‐GlcpN 1,4′‐bisphosphate (pentasaccharide bisphosphate) [Holst, O., Broer, W., Thomas‐Oates, J. E., Mamat, U. and Brade, H. (1993) Eur. J. Biochem. 214, 703–710]. The structures of the other two compounds were established by chemical analysis, NMR spectroscopy, and fast‐atom‐bombardment mass spectrometry as α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α‐D‐GlcpN 1‐phosphate (tetrasaccharide 1‐phosphate) and α‐Kdo‐(2–8)‐α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α‐D‐GlcpN 1‐phosphate (pentasaccharide 1‐phosphate).α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α/β‐D‐GlcpN 4′‐phosphate (pentasaccharide 4′‐phosphate) and α‐Kdo‐(2–8)‐α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α/β‐D‐GlcpN 4′‐phosphate (pentasaccharide 4′‐phosphate) were prepared from the 1,4′‐bisphosphates isolated from the recombinant strain Escherichia coli F515–207 by treatment with alkaline phosphatase and purification by high‐performance anion‐exchange chromatography and gel‐permeation chromatography. Their structures were characterised by chemical analysis, NMR spectroscopy, and fast‐atom‐bombardment mass spectrometry.
Quantification of two splicing events in the L‐type calcium channel α‐1 subunit of intestinal smooth muscle and other tissues
Authors: FERON Olivier , OCTAVE Jean‐Noël , CHRISTEN Marie‐Odile , GODFRAIND Théophile 
Published: March 2005

cDNA fragments encoding a representative region of the L‐type calcium channel α‐1 subunit of rabbit intestine smooth muscle were amplified by polymerase chain reaction (PCR). The nucleotide sequences of these intestine clones shared a high similarity with aorta, lung and heart calcium channels. However, in the extracellular loop between the third and fourth segments of domain IV and in the transmembrane IVS3 segment itself, we observed primary sequence variations corresponding to alternative splicing phenomenons.Since structural differences of L‐type calcium channel α‐1 subunits could result in functional variations, the respective expression frequency of these isoforms was determined in various tissues and species, and in the embryonic A7r5 cell line. The ontogeny of these splicing events was also examined from tissues of different ages.From this quantitative study, carried out by PCR of reverse‐transcribed mRNA, it clearly appears that the observed splicing processes in the IVS3–IVS4 region are not only tissue‐dependent but also regulated during development.
Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex
Authors: KASTRAU Dieter H. W. , HEISS Bernd , KRONECK Peter M. H. , ZUMFT Walter G. 
Published: March 2005

The nitric oxide reductase (NOR) from Pseudomonas stutzeri is a cytochrome bc complex which shows on SDS/PAGE two subunits with apparent molecular masses of 17 kDa and 38 kDa. Two other species of ∼45 kDa and 74–78 kDa represent the undissociated enzyme complex and an aggregate of the cytochrome b subunit, respectively. The cytochrome b subunit is highly hydrophobic and results in aberrant electrophoretic mobility. The stability of the enzyme in various detergents and at different pH was investigated. The highest specific activity of 60 μmol NO min−1 mg−1 protein was obtained after electrophoresis in the presence of laurylpropanediol‐3‐phosphorylcholine ether. Purified NOR contained cardiolipin, phosphatidylglycerol, and phosphatidylethanolamine, the latter as the major component. A phospholipid was required for high catalytic activity with either cardiolipin or phospatidylglycerol increasing the activity of the enzyme as isolated by a factor of up to 5. Free fatty acids inhibited NOR, with cis‐9‐octadecenoic acid (oleic acid) showing the most pronounced effect. Certain detergents substituted for the phospholipid requirement of NOR.The enzyme, as isolated, in 0.1% Triton X‐100, 20 mM Tris/HCl pH 8.5, exhibited a complex set of EPR resonances at low magnetic field, with a prominent peak at g 6.34 resulting from Fe(III) high‐spin cytochrome b. The second prominent feature arose from a low‐spin Fe(III) heme center with strong lines at apparent g values of 3.02 and 2.29, and a broad resonance at g∼ 1.5 which we assigned to the cytochrome c component of the enzyme. From spin quantitation and computer simulations of the various EPR signals a ratio close to 1:1 for the low‐spin/high‐spin heme centers in NOR was estimated. Shifting the pH from 8.5 to 5.0, replacing Triton X‐100 by other detergents, or adding soybean phospholipids to the protein, led to pronounced changes of the EPR signals in the g= 6 region. In contrast, the strong inhibitor oleic acid did not cause significant spectral changes. NOR which had been reduced by L‐ascorbate/phenazine methosulfate prior to incubation with its substrate NO gave the characteristic Fe(II) nitrosyl triplet centered at g∼ 2.01, with a hyperfine splitting of 1.70 mT. In the absence of dioxygen, NOR was quantitatively reduced by either sodium dithionite, or photochemically with deazaflavin and oxalate; the enzyme was reoxidizable by ferricyanide in a fully reversible reaction. Spectroelectrochemical oxidoreductive titrations gave E'0 (versus standard hydrogen electrode) =+322 mV for the cytochrome b and +280 mV for the cytochrome c component.
Kinetics of the reduction of wild‐type and mutant cytochromec‐550 by methylamine dehydrogenase and amicyanin from Thiobacillus versutus
Authors: UBBINK Marcellus , HUNT Nick I. , HILL H. Allen O. , CANTERS Gerard W. 
Published: March 2005

To elucidate the kinetic properties of the methylamine dehydrogenase (MADH) redox chain of Thiobacillus versutus the reduction of cytochrome c‐550 by MADH and amicyanin has been studied. Under steady state conditions, the rate constants of the reactions have been determined as a function of the ionic strength, both for wild type cytochrome c‐550 and for mutants in which the conserved residue Lys 14 has been replaced as follows: Lys14→Gln (mutant [K14Q]cytochrome c‐550) and Lys14→Glu (mutant [K14E]cytochrome c‐550).The second‐order rate constant of the reduction of cytochrome c‐550 by MADH shows a biphasic ionic‐strength dependence. At low ionic strength the rate constant remains unchanged (wild type) or increases ([K14Q]cytochrome c‐550) with increasing ionic strength, while at high salt concentrations the rate constant decreases monotonically as the ionic strength increases. It is suggested that conformational freedom exists in the association complex and that this is favourable for electron transfer. [K14Q]cytochrome c‐550 and [K14E]cytochrome c‐550 are reduced at rates 20‐fold and 500‐fold slower than wild‐type cytochrome c‐550 by MADH, due to a lower association constant. It is concluded that MADH possesses a negative patch with which cytochrome c‐550 associates. Lys14 plays an important role in the formation of the reaction complex.The midpoint potentials of wild‐type and mutant cytochrome c‐550 have been determined by using cyclic voltammetry. [K14Q]cytochrome c‐550 and [K14E]cytochrome c‐550 show an increase in E° of only 2 mV and 8 mV, respectively, compared to wild‐type cytochrome c‐550 (241 mV at pH 8.1).[K14Q]cytochrome c‐550 and [K14E]cytochrome c‐550 cytochrome c‐550 are reduced by amicyanin at rates that are only slightly faster than for wild‐type cytochrome c‐550. The difference is partly attributable to the change in E°. High ionic strength results in a threefold increase in the rate in all three cases. These results indicate that charge interactions do not play a major role in the formation of the amicyanin/cytochrome c‐550 reaction complex, suggesting an interaction at the hydrophobic patch of amicyanin.The reduction of cytochrome c‐550 by MADH can be inhibited by Zn2+‐substituted amicyanin. Ag+‐amicyanin, however, has little effect on the reduction rate. These results suggest that MADH has a much higher affinity for Cu2+‐amicyanin (substrate) than for Cu+‐amicyanin (product).On the basis of these findings the roles of the components of the MADH redox chain are discussed.
Site‐directed mutagenesis of cysteine residues in biliverdin reductase
Authors: MCCOUBREY William K. , MAINES Mahin D. 
Published: March 2005

Biliverdin reductase is unique among all enzymes described to date in having two pH optima, 6.75 and 8.7, at which NADH or NADPH, respectively, are required for activity. The enzyme converts biliverdin to bilirubin in mammals. The mature enzyme, which is 293 amino acids long, has 3 cysteine residues, and is sulfhydryl dependent. To understand the role of the cysteine residues in enzyme activity, we examined the effects of the neutral substitution with alanine of each of three residues, individually and in combination, by site‐directed mutagenesis. These residues in the predicted amino acid sequence of rat biliverdin reductase correspond to amino acids 73, 280 and 291. The modification of the amino‐proximal cysteine (Cys73), which is flanked by a tyrosine residue, completely inactivated the enzyme with NADH at pH 6.75 and NADPH at pH 8.7. The loss of reductase activity was not due to changes in three‐dimensional characteristics of the protein as suggested by its mobility in a non‐denaturing gel. Although modification of either of the two cysteines located near the C‐terminus (Cys280 and Cys291) significantly reduced activity with both cofactors, these mutations did not inactivate the enzyme. Comparison of Km values for the Cys280→Ala and Cys291→Ala mutants with the wild type protein, at pH 8.7, suggests that Cys280 principally functions in substrate binding while Cys291 is predominantly involved in cofactor binding. This assignment probably also applies at pH 6.75. Comparison of kcat of the mutants with wild type shows that mutation of Cys280 decreases Vmax of the enzyme. Mutation of both C‐terminal cysteines caused inactivation of the enzyme, comparable to that produced by mutation of Cys73. Analysis by circular dichroism at far‐ultraviolet wavelengths suggests that the alterations in activity are not the result of changes in the secondary structure of these mutants.These results are consistent with Cys73 having a central role in substrate/cofactor binding while biliverdin reductase can function, albeit at a reduced rate, with only one of the near C‐terminus cysteines. The results are further consistent with the suggestion that although the two C‐terminal cysteines have preferential affinities, they can serve similar functions in the interaction with substrate/cofactor.