bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthetic post‐translationally modified human Aβ peptide exhibits a markedly increased tendency to form β‐pleated sheets in vitro
Authors: FABIAN Heinz , SZENDREI Gyorgyi I. , MANTSCH Henry H. , GREENBERG Barry D. , ÖTVÖS Laszlo
Published: March 2005

The β‐amyloid peptide (Aβ) is the major constituent of senile plaques, one of the hallmark neuropathological lesions of Alzheimer's disease. Recently a post‐translationally modified analogue of the human β‐amyloid peptide, which contains isoaspartatic residues in positions 1 and 7, was isolated from parenchyma and leptomeningeal microvasculature of Alzheimer's disease patients [Roher, A. E., Lowenson, JD., Clarke, S., Wolkow, C., Wang, R., Cotter, R. J., Reardon, I. M., Zürcher‐Neely, H. A., Heinrikson, R. L., Ball, M. J. & Greenberg, B. D. (1993) J. Biol. Chem. 268, 3072–3083]. We used circular dichroism and Fourier‐transform infrared spectroscopy to characterize the conformational changes on human Aβ upon substitution of Asp1 and Asp7 to isoaspartic residues. We found that the intermolecular β‐pleated‐sheet content is markedly increased for the post‐translationally modified peptide compared to that in the corresponding unmodified human or rodent Aβ sequences both in aqueous solutions in the pH 7–12 range, and in membrane‐mimicking solvents (such as aqueous octyl‐β‐d‐glucoside or aqueous acetonitrile solutions). These findings underline the importance of the originally α‐helical N‐terminal regions of the unmodified Aβ peptides in defining its secondary structure and may offer an explanation for the selective aggregation and retention of the isomerized Aβ peptide in Alzheimer's‐disease‐affected brains.
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.
Multiple evolutionary origin of pyridoxal‐5′‐phosphate‐dependent amino acid decarboxylases
Authors: SANDMEIER Erika , HALE Terence I. , CHRISTEN Philipp
Published: March 2005

Comparison of the amino acid sequences of nine different pyridoxal‐5′‐phosphate‐dependent amino acid decarboxylases indicated that they can be subdivided into four different groups that seem to be evolutionarily unrelated to each other. Group I is represented by glycine decarboxylase, a component of a multienzyme system; group II comprises glutamate, histidine, tyrosine, and aromatic‐l‐amino‐acid decarboxylases; group III, procaryotic ornithine and lysine decarboxylase as well as the procaryotic biodegradative type of arginine decarboxylase; group IV, eucaryotic ornithine and arginine decarboxylase as well as the procaryotic biosynthetic type of arginine decarboxylase and diaminopimelate decarboxylase. (N–1) profile analysis, a more stringent application of profile analysis, established the homology among the enzymes of each group. A search with the profile of group II indicated a distant relationship with aminotransferases and thus with the α family of pyridoxal‐5′‐phosphate‐dependent enzymes. No evidence was obtained that groups I, III and IV were related with other pyridoxal‐5′‐phosphate‐dependent enzymes or any other protein in the databse. Unlike the aminotransferases, which, with few possible exceptions, costitute a single group of homologous proteins, the amino acid decarboxylases, by the criterion of profile analysis, have evolved along multiple lineages, in some cases even if they have the same substrate specificity.
Activity and fluorescence changes of lactate dehydrogenase induced by guanidine hydrochloride in reverse micelles
Authors: SHOSHANI Liora , DARSZON Alberto , TUENA DE GÓMEZ‐PUYOUM Marieta , GÓMEZ‐PUYOU Armando
Published: March 2005

Denaturants activate several multimeric enzymes in reverse micelles [Garza‐Ramos, G., Darszon, A., Tuena de Gómez‐Puyou, M. & Gómez‐Puyou, A. (1992) Eur. J. Biochem. 205, 509–517]. Here, the effect on activity and intrinsic fluorescence of pig heart lactate dehydrogenase (LDH) in reverse micelles [formed with 0.2 M cetyltrimethylammonium bromide in octane/hexanol (8.6:1, by vol.)] was explored at various water and guanidine hydrochloride (Gdn/HCl) concentrations. Emission fluorescence spectra of LDH in aqueous media and in micelles were similar. As in all aqueous media, 1.0 M Gdn/HCl in the water phase of reverse micelles produced fluorescence quenching and a blue shift of the maximal emission. In 5.0 M Gdn/HCl, instead of the red shift and significant quenching seen in water, the maximum emission further shifted to the blue and was only slightly quenched. Gdn/HCl titrations of activity and fluorescence changes of LDH in micelles with different water contents showed that at Wo ([H2O]/[surfactant]) of 6.6, 8.3, or 12.5, increasing concentrations of Gdn/HCl up to 0.6 M produced small changes in fluorescence, whereas activity increased several‐fold. At higher denaturant concentrations, activity decreased with significant fluorescence changes. In reverse micelles with 1 M Gdn/HCl, Vmax but not Km of LDH decreased with time. Under these conditions, there was progressive quenching of LDH fluorescence. The results show that in reverse micelles different Gdn/HCl concentrations induce variations in activity with or without alterations of the intrinsic fluorescence of LDH. The results also indicate that in reverse micelles, concentrations of Gdn/HCl below 1.0 M cause an enhancement of protein flexibility; this is accompanied by a marked increase in activity without important changes in intrinsic fluorescence. 1.0 M Gdn/HCl produces perturbations of inter‐subunit contacts that lead to fluorescence quenching and loss of catalytic activity, probably as consequence of dimerization of tetrameric LDH.
Purification and characterization of two forms of soluble thrombomodulin from human urine
Authors: JACKSON Denise E. , TETAZ Tim J. , SALEM Hatem H. , MITCHELL Christina A.
Published: March 2005

We have isolated and characterized two forms of soluble thrombomodulin from human urine. The purification procedure consisted of ultrafiltration, chromatography on DEAE‐Sepharose, affinity chromatography on diisopropyl‐phosphate–thrombin and/or monoclonal anti‐thrombomodulin IgG affigel followed by reverse‐phase HPLC. An active soluble form of thrombomodulin was purified 1600‐fold from 34‐1 urine. The purified protein migrated as a doublet, with molecular mass 76/72 kDa under reducing conditions and 63/57 kDa under non‐reducing conditions as determined by SDS/PAGE. Amino acid analysis of the 63/57‐kDa soluble thrombomodulin confirmed sequence identity with human thrombomodulin and demonstrated N‐terminal heterogeneity. Compared to membrane‐type thrombomodulin, the purified 63/57‐kDa soluble thrombomodulin was more active as a cofactor for protein‐C activation. The second major thrombomodulin fragment in urine is an inactive 35‐kDa thrombomodulin polypeptide derived from the N‐terminal extracellular region of thrombomodulin.
Identification of tissue‐specific isoforms for subunits Vb and VIIa of cytochrome c oxidase isolated from rainbow trout
Authors: FREUND Reinhard , KADENBACH Bernhard
Published: March 2005

Cytochrome c oxidase was isolated from heart and liver of rainbow trout (Salmo gairdnerii). SDS/PAGE analysis showed the presence of 11 different polypeptide subunits in the fish enzyme. The nuclear‐coded subunits IV, Va, Vb, VIc, VIIa, VIIc and VIII could be identified by their N‐terminal amino acid sequences. The mammalian subunits VIa and VIlb appear to be absent (or blocked at the N‐terminal) in cytochrome c oxidase from trout. For subunit Vb, two polypeptides of different electrophoretic mobilities were found which differed in their N‐terminal sequences, and represent a new pair of cytochrome‐c‐oxidase subunit isoforms, not found in mammalia. Both isoforms of subunit Vb were found in cytochrome c oxidase from heart and liver, but at different ratios. Subunit VIIa also seemed to occur in different isoforms, whereas subunit VIII had the same N‐terminal amino acid sequence in cytochrome c oxidase of liver and heart, similar to the human‐type subunit but different from rat, bovine and chicken.
The NAM1 protein (NAM1p), which is selectively required for cox1, cytb and atp6 transcript processing/stabilisation, is located in the yeast mitochondrial matrix
Authors: WALLIS Mary G. , GROUDINSKY Olga , SLONIMSKI Piotr P. , DUJARDIN Geneviève
Published: March 2005

The NAM1 nuclear gene was shown to control the stability and/or processing of mitochondrial transcripts of the cytochrome b, cytochrome oxidase subunit I and ATP synthase subunit VI genes [Groudinsky O., Bousquet I., Wallis M. G., Slonimski P. P. & Dujardin G. (1993) Mol. Gen. Genet. 240, 419–427]. In order to better understand the mode of action of the NAM1 gene product, we have examined its intracellular fate. A fusion plasmid enabling bacterial over‐expression of the corresponding protein‐A–NAM1 cognate was constructed and subsequently employed as an antigen to raise polyclonal antibodies. These antibodies specifically recognise a 50‐kDa protein which purifies along with the mitochondria and corresponds to NAM1p. Submitochondrial localisation experiments show that NAM1p is a soluble protein, located interior to the mitoplasts. Matricial location is a strong argument in favour of a direct interaction of NAM1p with particular mitochondrial transcripts and leads us to propose a model in which NAM1p could be an RNA‐convoying protein stabilising and directing mitochondrial transcripts towards the inner face of the inner membrane where translation and assembly seem to occur.
Procarboxypeptidase in rat pancreas Overall characterization and comparison of the activation processes
Authors: OPPEZZO Oscar , VENTURA Salvador , BERGMAN Tomas , VENDRELL Josep , JÖRNVALL Hans , AVILÉS Francesc X.
Published: March 2005

Three monomeric procarboxypeptidases and a binary complex consisting of a procarboxypeptidase and a chymotrypsinogen have been isolated from rat pancreas by HPLC. N‐terminal sequence determination, substrate‐specificity analysis and physico‐chemical characterization showed that the carboxypeptidase precursors were the A1, A2 and B forms. No isomorphism could be detected for any of these proenzymes and no clear evidence was obtained for the presence of procarboxypeptidase‐containing quaternary complexes of the types previously described for other species. Instead, we observed the presence of a binary complex between procarboxypeptidase A2 and chymotrypsinogen B. Among the major pancreatic endoproteinases, only trypsin was found to be a general activator of rat procarboxypeptidases in vitro. Time‐course analysis of the products generated after trypsin addition confirmed that full activation of procarboxypeptidase A1 requires several cleavages in the C‐terminal region (residues 87–94) of the activation segment, while procarboxypeptidases A2 and B require a single cleavage each. The carboxypeptidases released participate in the trimming of the activation segment in A1 and B, but not in A2, probably because of the high specificity of the latter in the active form.
Mapping of Hsp70‐binding sites on protein antigens
Authors: ROMÁN Eulogia , MORENO Carlos , YOUNG Douglas
Published: March 2005

Hsp70‐binding sites were mapped on three antigens, the 16‐, 19‐ and 38‐kDa proteins of Myco‐bacterium tuberculosis, using overlapping synthetic peptides in a competitive‐binding assay. In each protein, two or three prominent hsp70‐binding sites were identified when peptides 20‐amino‐acid long were used, predominantly in regions containing clusters of aliphatic amino acids. Although there was an overall concordance in the pattern of peptide binding to hsp70 from bacterial (M. tuberculosis) and mammalian sources (immunoglobulin heavy‐chain‐binding protein), some differences in the specificity of polypeptide binding and the effect of peptides on ATPase activity were observed.
The process of cellular uptake of iron from transferrin
Authors: BAKØY Ole E. , THORSTENSEN Ketil
Published: March 2005

In an attempt to improve our understanding of the complex interplay between cell compartments and chemical species during cellular uptake of iron from transferrin, we designed a computer simulation program based on current models of receptor‐mediated endocytosis and pinocytosis. The program calculates and visualizes, as a function of time, the changes in transferrin, apotransferrin, and iron concentrations occurring in all relevant cellular compartments during cellular iron acquisition from transferrin. Simulation of literature data showed that the program generates results that are in accordance with experimental data. Furthermore, from measurements of the uptake of [carboxyl‐14C]dextran we could utilize the program to suggest rate constants characteristic for the pinocytic process in rat reticulocytes. Moreover, simulations indicate that the apparent difference in the iron uptake process observed between reticulocytes and hepatocytes may be explained by the contribution made by pinocytosis to the iron uptake process. Finally, the present program should have potential as an educational tool during introduction to the field of receptor‐mediated endocytosis in general and to cellular iron metabolism in particular.
Two functionally distinct half sites in the DNA‐recognition sequence of the Myb oncoprotein
Authors: ORDING Eli , KVÅVIK Wenche , BOSTAD Anne , GABRIELSEN Odd S.
Published: March 2005

The oncoprotein Myb is a sequence‐specific DNA‐binding protein with a pivotal function in the development and proliferation of hematopoietic precursor cells. A minimal DNA‐binding domain composed of two tryptophan‐rich repeats R2 and R3 is responsible for sequence recognition. Based on model building and mutational analysis, Myb was proposed to recognise its target through a double helix‐turn‐helix (HTH)‐related motif using two recognition helices, one in R2 and one in R3. We found, by mutational analysis, that the DNA‐binding site for c‐Myb is functionally bipartite. While the first half site is dominant and absolutely required for binding, the second half site is only modulatory and mainly affects the half life of the complex. This bipartite nature of the binding site parallels the proposed bipartite structure of R2R3 with two HTH‐related domains. Analysis of the DNA‐binding site of R2R3 by missing‐base interference‐footprint analysis showed that the protein interacted with a 9‐bp region. The same was found with a larger protein containing all three repeats. The effect of adding R1 was mainly to stabilise the complex. The borders of the complex, as revealed by exonuclease III footprinting, did not change due to the presence of R1. However, both borders became more refractory to the nuclease when R1 was present, but with a difference that suggested a specific orientation of the repeat domains relative to the DNA‐binding site. We propose that the first half site is recognised by R3, while the second modulatory half site interacts with the R2 repeat.
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.
Solid‐phase synthesis, conformational analysis and in vitro cleavage of synthetic human synaptobrevin II 1–93 by tetanus toxin L chain
Authors: CORNILLE Fabrice , GOUDREAU Nathalie , FICHEUX Damien , NIEMANN Heiner , ROQUES Bernard P.
Published: March 2005

A 93‐residue peptide corresponding to the cytosolic domain of a human vesicle associated membrane protein (VAMP or synaptobrevin) has been prepared by solid‐phase peptide synthesis in order to investigate the proteolytic activity of the tetanus toxin light chain (TeTx L chain). This protein has been recently reported to inactivate the neuronal rat synaptobrevin II by proteolysis. We show in this study that the synthetic human synaptobrevin II 1–93 (Syb II 1–93) as well as an N‐terminus‐shortened 69‐residue peptide (Syb II 25–93) were cleaved selectively at the Gln76‐Phe77 peptide bond by TeTx L chain while shorter peptides were not. A Michaelis constant Km= 192 ± 2 μM and a catalytic constant kcat= 0.5 min−1 were found for the 93‐residue peptide. A neutral optimum pH for the cleavage rate, an inhibition by preincubation of the toxin with well known nonspecific inhibitors of metallopeptidases as well as a zinc‐dependent enzyme activity suggest that TeTx belongs to the zinc endopeptidase family. Moreover an activation by reducing agents and an inhibition by cysteine‐modifying chemical reagents indicate a critical thiol dependency. Among several specific inhibitors of zinc endopeptidases tested, none could inhibit TeTx L chain even at high concentration. Structural studies by 600‐MHz 1H‐NMR showed that in water or dimethyl‐sulfoxide the peptide Syb II 1–93 and shorter fragments did not present well defined conformations. Nevertheless protein–protein interactions have been shown for the peptides Syb II 1–93 and 25–93 but not for Syb II 51–93, a fragment not cleaved by TeTx L chain.
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.
Protein purification, gene cloning and sequencing of an acidic endoprotease from Myxococcus xanthus DK101
Authors: LUCAS Nathalie , MAZAUD‐AUJARD Catherine , BREMAUD Laure , CENATIEMPO Yves , JULIEN Raymond
Published: March 2005

An acidic endoprotease (MAEP) secreted during vegetative growth by Myxococcus xanthus DK101 was purified to homogeneity by a series of chromatographic procedures. The endoprotease cleaved the Phe‐Met bond of K‐casein under acidic conditions (pH 5.9). Its apparent molecular mass and its isoelectric point have been estimated to be 12 kDa and 4.5, respectively. From the N‐terminal amino acid sequence, a set of two primers for polymerase chain reaction have been designed. Amplification of the corresponding DNA fragment (84 bp) generated a probe, then used to screen an expression DNA library of M. xanthus and to isolate a recombinant plasmid which contained a 2127‐bp insert. The nucleotide sequence included an open reading frame (ORF) of 585 nucleotides, encoding 195 amino acids, that exhibited a high degree of similarity with the N‐terminal amino acid sequence of the purified MAEP. The polypeptide sequence inferred from this ORF revealed that the mature enzyme should contain 131 amino acids arising from a 195‐amino‐acid precursor protein.
Streptokinase activates plasminogen bound to human group C and G streptococci through M‐like proteins
Authors: BEN NASR Abdelhakim , WISTEDT Annika , RINGDAHL Ulrika , SJÖBRING Ulf
Published: March 2005

An ability to interact with plasminogen or plasmin could provide micro‐organisms with a mechanism for invasion. Thus, group A, C and G streptococci secrete streptokinase which binds and activates plasminogen. Some streptococci also express surface structures which bind plasminogen without causing its activation. Plasminogen‐binding surface proteins were extracted from one group C and one group G streptococcal isolate. Both proteins were found to bind plasmin, fibrinogen and serum albumin in addition to plasminogen. Gene fragments encoding the streptococcal proteins were amplified by PCR and were subsequently cloned and expressed in Escherichia coli. DNA sequence determination revealed for both genes open reading frames encoding proteins which contained repetitive domains and a carboxyl‐terminal unrepeated region that were typical of M and M‐like proteins. Though the amino‐terminal regions of the group C and G streptococcal proteins demonstrated a rather high overall similarity between themselves, they were not similar to the variable regions of other M‐like proteins with one exception: there was a 46% identity between the first 22 amino acids of the group G streptococcal protein and the corresponding sequence of PAM, the plasminogen‐binding M‐like protein of type M53 group A streptococci. Like the proteins extracted from the streptococci, the recombinant proteins bound plasminogen, fibrinogen and albumin. The three plasma proteins bound to separate sites on the streptococcal M‐like proteins. Plasminogen bound by the group C and G streptococcal proteins was readily activated by streptokinase, providing evidence for a functional link between the secreted plasminogen‐activator and proteins exposed on the bacterial surface.
Purification and characterization of chaperonin 60 and chaperonin 10 from the anaerobic thermophile Thermoanaerobacter brockii
Authors: TRUSCOTT Kaye N. , HØJ Peter B. , SCOPES Robert K.
Published: March 2005

Chaperonin 60 and chaperonin 10 (GroEL and GroES homologues, respectively) have been isolated from extracts of the anaerobic thermophile Thermoanaerobacter brockii. A simple and rapid purification for chaperonin 60 made use of hydrophobic and anion‐exchange chromatographies, and could be readily scaled up; approximately 2 mg pure chaperonin 60 was obtained/g cells. In contrast with all other prokaryotic chaperonin 60 proteins that have been studied, which are tetradecamers, including those from Thermus sp., the T. brockii protein is a heptamer, and as isolated was not in association with chaperonin 10. The preparation is readily crystallized using 2‐propanol or poly(ethylene glycol) with MgCl2. The N‐terminal amino acid sequence of this preparation is similar to other thermophilic chaperonin 60 proteins. Chaperonin 10 was purified from the flow‐through of the first hydrophobic column (which bound chaperonin 60) using a more hydrophobic adsorbent to remove contaminating proteins, followed by anion‐exchange chromatography. Chaperonin 10 was obtained with a yield of approximately 10% that of chaperonin 60. The subunit molecular mass of chaperonin 10 determined by electrospray mass spectrometry is 10254 ± 0.4Da, which is very similar to the molecular mass of Escherichia coli GroES. Similarly, the subunit size of chaperonin 60 determined by mass spectrometry is very similar to that of GroEL, at 57949 ± 10Da. T. brockii chaperonin 60 has an ATPase activity that is suppressed by chaperonin 10, and the two proteins together are active in protein‐folding assays. Mitochondrial malate dehydrogenase was successfully refolded at 37°C after denaturation in guanidine hydrochloride, using T. brockii chaperonin 60 and chaperonin 10, or chaperonin 60 and E. coli GroES. The denatured enzyme was protected from aggregation by association with chaperonin 60. Guanidine‐hydrochloride‐denatured preparations of isocitrate dehydrogenase and secondary alcohol dehydrogenase isolated from T. brockii were also refolded at 60–65°C. In each case, refolding required chaperonin 60, chaperonin 10 and ATP, giving up to 80% regeneration of control activity.
Interfacial kinetic reaction of human 5‐lipoxygenase
Authors: NOGUCHI Masato , MIYANO Masashi , KUHARA Satoru , MATSUMOTO Takashi , NOMA Masana
Published: March 2005

The kinetics of human 5‐lipoxygenase were investigated in the presence of Tween 20 using a continuous spectrophotometric assay. Using the mixture at a constant molar ratio of arachidonate/Tween 20 at pH 8.0, the steady‐state velocity on a varied arachidonate concentration did not follow simple Michaelis‐Menten‐type kinetics and double‐reciprocal plot analysis gave hyperbolic curves. However, by introducing the concept of a local pH change, it was possible to analyze the kinetics as simple Michaelis‐Menten type. The concept of a local pH change implies that when utilizing an acidic and amphiphilic substance as a substrate, such as arachidonate, the medium around the substrate is acidified with an increased concentration of substrate. This concept was explained rationally by two experiments. Consequently, the data were transformed according to a local pH change and analyzed according to a dual phospholipid model as has been proposed for phospholipase A2 [Hendrickson, H. S. and Dennis, E. A. (1984) Kinetic analysis of the dual phospholipid model for phosphalipase A2, J. Biol. Chem. 259, 5734–5739]. It is concluded that 5‐lipoxygenase performs an interfacial reaction in the arachidonate/Tween 20 mixed micelles in the same manner as phospholipase A2. The values of Km were almost constant (about 0.07 molar fraction), even when arachidonate molar ratios were changed in the surface of the mixed micelles. The values for Ks (the association constant of the enzyme to the micelle interface) ranged over 0.21–0.48 μM. The Vmax was 25.76 μmol · min−1· mg−1. This concept of a local pH change could be used extensively with enzymes which utilize both amphiphilic and acidic substances as substrates.
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.
New non‐lethal calmodulin mutations in Paramecium
Authors: LING Kit‐Yin , MALEY Margot E. , PRESTON Robin R. , SAIMI Yoshiro , KUNG Ching
Published: March 2005

The mechanisms by which calmodulin coordinates its numerous molecular targets in living cells remain largely unknown. To further understand how this pivotal Ca2+‐binding protein functions in vivo, we isolated and studied nine new Paramecium behavioral mutants defective in calmodulin. Nucleotide sequences of mutant calmodulin genes indicated single amino‐acid substitutions in mutants cam4(E104K), cam5‐1 (D95G), cam6 (A102V), cam7 (H135R), cam14‐1 (G59S) and cam15 (D50G). In addition, we encountered a second occurrence of three identified substitutions; they are cam1‐2 (S101F), cam5‐2 (D95G) and cam14‐2 (G59S). Most of these mutational changes occurred in sites that have been highly conserved throughout evolution. Furthermore, most of these changes were not among the amino acids known to interact with the basic amphiphilic peptides of calmodulin targets. Consistent with our previous finding [Kink, J. A., Maley, M. E., Preston R. R., Ling, K.‐Y., Wallen‐Friedman, M. A., Saimi, Y. & Kung, C. (1990) Cell 62, 165–174], mutants that under‐reacted to certain stimuli (allele number above 10) had substitutions in the N‐terminal lobe of calmodulin, and those that over‐reacted (below 10) had substitutions in the C‐terminal lobe. No mutations were found in the central helix that connects the lobes. Thus, through undirected in vivo mutation analyses of Paramecium, we discovered that each of the two lobes of calmodulin has a distinct role in regulating the function of a specific ion channel and eventually the behavior of Paramecium. We, therefore, propose a hypothesis of functional bipartition of calmodulin that reflects its structural bipartition.
Determination of membrane‐bound fragments of cytochrome P‐450 2B4
Authors: UVAROV Valentin Yu. , SOTNICHENKO Alexander I. , VODOVOZOVA Elena L. , MOLOTKOVSKY Julian G. , KOLESANOVA Ekaterina F. , LYULKIN Yuri A. , STIER Antony , KRUEGER Volker , ARCHAKOV Alexander I.
Published: March 2005

Membrane‐bound sites of cytochrome P‐450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar ‘head’ were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N‐terminal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N‐terminus but also to the segments 273–314 and 427–491. Epitope mapping of the domain next to the N‐terminus (residues 21–119) of the isolated hemoprotein was performed with the help of a peptide‐scanning method, a programmable peptide synthesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P‐450 2B4. This domain was shown to possess a considerable density of sites with high antigenic activity. No membrane‐penetrating part of this domain was found except for the fragment 1–21. A model of structure of P‐450 2B4 was computed by comparison with the structure of cytochrome P‐450cam on the basis of an alignment of 47 cytochromes P‐450 with the former hemoprotein. Major parts of the protein sequences photoreacting with the phospholipid probes, but not the antibody‐reactive epitopes of the region 21–119, are located at the membrane‐facing side in this model
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.