bluuebunny/crossref_metadata_2025 huggingface dataset:
5′‐Methylbenzimidazolyl‐cobamides are the corrinoids from some sulfate‐reducing and sulfur‐metabolizing bacteria
Authors: KRÄUTLER Bernhard , KOHLER Hans‐Peter E. , STUPPERICH Erhard
Published: March 2005

The sulfate‐reducing bacteria Desulfobacterium autotrophicum, Desulfobulbus propionicus and Archaeoglobus fulgidus (VC‐16) and the sulfur‐metabolizing archaebacteria Desulfurolobus ambivalens and Thermoplasma acidophilum were found to contain considerable amounts of corrinoids, that were isolated and crystallized in their Coβ‐cyano form. In three other sulfur‐metabolizing archaebacteria, Thermoproteus neutrophilus, Pyrodictium occultum and Staphylothermus marinus significant amounts of corrinoids were not detected under the isolation methods used.

The samples from the three sulfate‐reducers were identified with Coα‐[α‐(5′‐methylbenzimidazolyl)]‐Coβ‐cyanocobamide. This corrinoid was also obtained from a 5‐methylbenzimidazole‐supplemented Propionibacterium fermentation and was structurally characterized by ultraviolet/visible, CD, fast‐atom‐bombardment MS, 1H‐ and 13C‐NMR spectroscopy. Also the major corrinoid from T. acidophilum was (tentatively) analyzed as a 5′‐methylbenzimidazolyl‐cobamide, whereas the main corrinoid from D. ambivalens was indicated to be vitamin B12 (a 5′,6′‐dimethylbenzimidazolyl‐cobamide).

The 5′‐methylbenzimidazolylcobamides are found here as the common corrins of some sulfate‐reducing and sulfur‐metabolizing bacteria. The structural diversity due to the differing nucleotide bases of the corrins examined here and in methanogenic and acetogenic bacteria appears not to correlate to the biological function(s) of the corrins, but rather to be determined by biosynthetic properties of these organisms under natural growth conditions.
Characterization of the cross‐reacting determinant (CRD) of the glycosyl‐phosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoprotein
Authors: ZAMZE Susanne E. , FERGUSON Michael A. J. , COLLINS Ruth , DWEK Raymond A. , RADEMACHER Thoma W.
Published: March 2005

The cross‐reacting determinant (CRD epitope) of the glycosyl‐phosphatidylinositol (GPI) membrane anchor of Tryponosoma brucei glycoprotein has been analysed by selective chemical and enzymic modification of the isolated GPI structure combined with the use of a competitive ELISA inhibition assay for the detection of CRD epitopes. The data show that the CRD consists of at least three overlapping epitopes involving different regions of the molecule including the inositol 1, 2‐cyclic phosphate, the non‐N‐acetylated‐glucosamine residue and the galactose branch. Although the presence of all three of these structural features is required for quantitative binding of anti‐CRD antibodies in ELISA and Western blotting, the Western blot reaction obtained inthe presence of any one epitope is still significant. The use of anti‐CRD antibodies for the detection of GPI anchors is discussed.
Analysis of the activation of pepsinogen in the presence of protein substrates and estimation of the intrinsic proteolytic activity of pepsinogen
Authors: KAGEYAMA Takashi
Published: March 2005

Monkey pepsinogen A, monkey progastricsin, and porcine pepsinogen A were activated in the presence of two different protein substrates, namely, reduced and carboxymethylated lysozyme and hemoglobin. In each case, an extensive delay in activation was observed. The intermolecular activation reaction required for the generation of pepsin or gastricsin was strongly inhibited and this inhibition was essentially responsible for the delay. However, the intramolecular reaction required for the generation of the intermediate forms of the proenzymes was scarcely affected. The delay was longer at pH 3.0 than at pH 2.0.

Irrespective of the delay in activation of pepsinogen, the digestion of substrates proceeded rapidly, evidence of the significant proteolytic activity of pepsinogen itself. Kinetic experiments demonstrated that pepsinogen changed from an enzymatically inactive species to an active species before the release of the activation segment. The proteolytic activity of the active pepsinogen was highest at pH 2.0, at 37°C and the activity under these conditions was comparable to that of pepsin.
The subunit structure of a major glutathione S‐transferase form, MT, in rat testis
Authors: ISHIKAWA Takashi , TSUCHIDA Shigeki , SATOH Kimihiko , SATO Kiyomi
Published: March 2005

A major glutathione S‐transferase form (pI 5.7) in rat testis (MT) purified by S‐hexyl‐glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pI 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two‐dimensional gel electrophoresis. Rechromatofocusing o the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4). These were identified as the two homodimers consisting of the subunits of MT, Yn1 Yn1 and Yn2 Yn2 respectively. Furthermore, MT could be reconstituted from Yn1 Yn1 and Yn2 Yn2. These results indicate that MT is a heterodimer, Yn1 Yn2, consisting of subunits with very similar molecular masses but different isoeleetric points. The Yn1 Yn1 form had glutathione S‐transferase activities towards l‐chloro‐2,4‐dinitrobenzene and 1,2 dichloro‐4‐nitrobenzene. However, the Yn2 Yn2 form had no activity towards any of the substrates examined N‐terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1 and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N‐terminal amino acid sequences. Subunits Ynl and Yn2 seem to belong to the rat GST 3‐4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1 Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159‐164]. The Yn2 Yn2 form seemed to differ from GST 5‐5 and may be a new form of rat glutathione S‐transferase.
Microtubule‐assembly inhibitor protein
Authors: KOTANI Susumu , IKAI Atsushi , KAWAI Gota , MAEKAWA Shohei , YOKOYAMA Shigeyuki , SAKAI Hikoichi
Published: March 2005

Microtubule‐assembly inhibitor protein (MIP) is an acidic protein with Mr 33000 which inhibits microtubule assembly in vitro [Kotani, S., Murofiushi, H., Nishida, E. & Sakai, H. (1984) J. Biochem. (Tokyo) 96, 959‐969]. Anti‐MIP antibody was affinity‐purified from rabbit anti‐MIP sera raised against chemically modified MIP. MIP was localized in the nucleus in interphase culture cells as revealed by immunofluorescent light microscopy.

Immunoblotting experiments showed that MIP exists in a variety of mammalian cells and tissues. Kidney appeared to be a better source of MIP than brain, the original source. Kidney MIP was isolated by the same procedure as for brain MIP and proved to be indistinguishable from brain MIP in the inhibitory acitivity of microtubule assembly, molecular mass, immunoreactivity, and one‐dimensional peptide mapping. Physicochemical characteristics of MIP were studied using the kideny protein. It contained 20% aspartic acid and 25% glutamic acid, accounting for its acidic nature. Hydrodynamically, MIP was a monomer with s20, w= 1.9 S and Mr= 30 000. The frictional ratio, f f0= 1.7, indicated that MIP is not a glubular molecule but has either an elongated or an expanded structure. Circular dichroic results showed a low content of α‐helix or β‐sheet structure for MIP. Proton nuclear magnetic resonance analysis provided evidence that MIP consists mainly of very flexible structures (random‐coli‐like structures), but still contains a hydrophobic core structure below 60°C.
Trichokirin, a ribosome‐inactivating protein from the seeds of Trichosanthes kirilowii Maximowicz
Authors: CASELLAS Pierre , DUSSOSSOY Danielle , FALASCA Anna Ida , BARBIERI Luigi , GUILLEMOT Jean Claude , FERRARA Pascual , BOLOGNESI Andrea , CENINI Pietro , STIRPE Fiorenzo
Published: March 2005

A protein, here named trichokirin, was extracted from the seeds of Trichosanthes kirilowii and purified by ion‐exchange and gel‐filtration chromatography. Trichokirin is a basic glycoprotein of apparent relative molecular mass of 27000 with a strong ribosome‐inactivating activity. Alignment of the trichokirin, trichosanthin and momordin N‐terminal sequences shows a substantial degree of homology. Trichokirin was conjugated to a monoclonal antibody directed against the Thy 1.2 antigen with the cleavable dimethyl 3,3′‐dithiobispropionimidate cross‐linking reagent. This immunotoxin selectively killed leukemia cells expressing the Thy 1.2 antigen. The addition of ammonium chloride, which increases the cytotoxicity of ricin A‐chain immunotoxins, blocks that of the trichokirin immunotoxin, suggesting that they enter cells by different mechanisms. In vivo studies showed that the pharmacokinetic properties of the trichokirin immunotoxin could be more advantageous than those of the ricin A‐chain immunotoxins for in vivo applications.
DNA‐binding properties of gene‐5 protein encoded by bacteriophage M13
Authors: BULSINK Henk , HARMSEN Ben J. M. , HILBERS Cornelis W.
Published: March 2005

The binding of gene‐5 protein, encoded by bacteriophage M13, to oligodeoxynucleic acids was studied by means of fluorescence binding experiments, fluorescence depolarization measurements and irreversible dissociation kinetics of the protein · nucleotide complexes with salt. The binding properties thus obtained are compared with those of the binding to polynucleotides, especially at very low salt concentration. It appears that the binding to oligonucleotides is always characterized by a stoichiometry (n) of 2‐3 nucleotides/protein, and the absence of cooperativity. In contrast the protein can bind to polynucleotides in two different modes, one with a stoichiometry of n= 3 in the absence of salt and another with n= 4 at moderate salt concentrations. Both modes have a high intramode cooperativity (ω about 500) but are non‐interacting and mutually exclusive. For deoxynucleic acids with a chain length of 25‐30 residues a transition from oligonucleotide to polynucleotide binding is observed at increasing nucleotide/protein ratio in the solution. The n= 3 polynucleotide binding is very sensitive to the ionic strength and is only detectable at very low salt concentrations. The ionic strength dependency per nucleotide of the n= 4 binding is much less and is comparable with the salt dependency of the oligonucleotide binding. Furthermore it appears that the influence of the salt concentration on the oligonucleotide binding constant is to about the same degree determined by the effect of salt on the association and dissociation rate constants. Model calculations indicate that the fluorescence depolarization titration curves can only be explained by a model for oligonucleotide binding in which a protein dimer binds with its two dimer halves to the same strand. In addition it is only possible to explain the observed effect of the chain length of the oligonucleotide on both the apparent binding constant and the dissociation rate by assuming the existence of interactions between protein dimers bound to different strands. This results in the formation of a complex consisting of two nucleotide strands with protein in between and stabilized by the dimer‐dimer interactions.
The arrangement of intra‐ and intermolecular disulfide bonds in the carboxyterminal, non‐collagenous aggregation and cross‐linking domain of basement‐membrane type IV collagen
Authors: SIEBOLD Bernhard , DEUTZMANN Rainer , KÜHN Klaus
Published: March 2005

The hexameric complex of globular domains of type IV collagen was isolated after collagenase digestion of human placenta and the different monomers and dimers present were chromatographically separated. The ratio of α1(IV)NC1 to α2(IV)NC1 was 2:1. About 50% of the NC1 domains were connected to dimers. Predominantly α1‐α1 dimers were found. Only 12% were α2‐α2 dimers and no α1‐α2 dimers could be detected. The majority (88%) of the intermolecular bonds was found to be disulfide bridges. The remainder could not be cleaved by reduction.

To elucidate the arrangement of the disulfide bonds, the unreduced α(IV)NC1 monomers were treated with cyanogen bromide, the disulfide‐bridged peptides isolated and characterized by Edman degradation. Each of the two homologous subdomains within a monomer is stabilized by an identical set of three disulfide bonds. In subdomain I, cysteines at positions 20 and 53 are connected with the C‐terminal cysteine pair 108 and 111. Thus formed, the disulfide knot stabilizes two interconnected loops of 32 and 54 residues, respectively. A smaller loop of five residues occurs due to a disulfide bond between the cysteines 65 and 71. A similar disulfide arrangement is indicated for subdomain II which is separated from subdomain I by a segment of 20 amino acid residues. The same arrangement of disulfide bonds has been strongly suggested for the α2(IV)NC1 monomer by the isolation and characterization of its disulfide‐bridged tryptic fragments.

Similar investigations on the dimeric α1(IV)NC1 domain established the arrangement of the intermolecular disulfide bonds. They are formed by a complete disulfide exchange between corresponding disulfide knots of two monomeric NC1 domains.
p‐Benzylthiocarbamoyl‐aspartyl‐daunorubicin‐substituted polytrisacryl
Authors: DAUSSIN François , BOSCHETTI Egisto , DELMOTTE Francis , MONSIGNY Michel
Published: March 2005

A new type of drug acid‐labile arm‐carrier is described. A hydrophilic polymer [polytrisacryl, or poly(acryloyl 2‐amido‐2‐hydroxymethyl‐1,3‐propanediol)], used as a model, is substituted with p‐nitrobenzyl groups. The nitrobenzyl groups are reduced to aminobenzyl and transformed into benzylisothiocyanate groups which are allowed to react with aminoacyl daunorubicin. The excess of benzylisothiocyanate is transformed into benzylthiocarbamoyl N‐methyl glucamine. A conjugate containing benzylthiocarbamoyl‐aspartyl daunorubicin is stable at neutral pH, and releases free daunorubicin when it is exposed to pH 5 or below. This conjugate is about 200‐fold less toxic than free daunorubicin, on a molecular basis, when it is added to the medium of Lewis lung carcinoma (3LL) cells in culture.
Interaction of heparin and antithrombin III
Authors: PETITOU Maurice , LORMEAU Jean‐Claude , CHOAY Jean
Published: March 2005

A synthetic pentasaccharide corresponding to the sequence involved in heparin for binding and activation of antithrombin III contains eight sulfate groups. The role of some of them in the interaction with the protein has been demonstrated through the study of fragments obtained from heparin. An approach based on the total chemical synthesis of heparin fragments allows us to provide new information on the O‐sulfate groups borne by the iduronic acid and the glucosamine units that constitute the reducing‐end disaccharide of the above pentasaccharide sequence. Although not strictly necessary for a weak interaction to take place, these two sulfates co‐operate to express maximal activity. This suggests that they belong to a secondary sub‐region of interaction with antithrombin III, the primary one being accounted for by other critical parts of the structure and particularly the trisaccharide sequence placed at the non‐reducing end of the pentasaccharide.
Methoxyretinals in bacteriorhodopsin
Authors: GÄRTNER Wolfgang , OESTERHELT Dieter
Published: March 2005

Analogue bacteriorhodopsins (BRs) were reconstituted from bacterioopsin and 9‐, 11‐, or 13‐methoxyretinals or their demethyl derivatives, respectively. In organic solvents the retinals occur as cis isomers of the respective double bonds carrying the methoxy group. 9‐Methoxyretinal, present as the 9‐cis isomer, does not form an analogue BR with bacterioopsin in the dark. Upon illumination, a BR is produced with an absorbance maximum at 560 nm. This compound is thermally unstable, and converts back into the 9‐cis‐containing complex (λmax= 410 nm) in the dark. Removal of the 13‐methyl group from this compound (= 9‐methoxy 13‐demethyl retinal) does not change the 9‐cis configuration of the free retinal, but allows the reconstitution of a thermally stable chromoprotein absorbing around 500 nm with a proton translocation rate of about 10% of the BR value, comparable to the 13‐demethyl BR value [Gärtner, W., Towner, P., Hopf, H. & Oesterhelt, D. (1983) Biochemistry 22, 2637‐2644]. 11‐Methoxy BRs (13‐demethyl and 9,13‐didemethyl) absorb around 530 nm and are inactive. 13‐Methoxy retinal (13‐cis isomer) reconstitutes a chromoprotein with an absorbance maximum at 515 nm, which can be photoconverted to a thermostable 460‐nm‐absorbing complex. For the 515‐nm‐absorbing species of 13‐methoxy BR a light‐induced proton translocation was not detected in measurements with cell vesicles (detection of pH changes in the vesicle preparation). Only by photocurrent measurements in a bilayer experiment could a very diminished photocurrent be detected, about 1 ‐ 2% of BR, [Fendler et al. (1987) Biochim. Biophys. Acta 893, 60‐68]. The reconstitution rate of 13‐methoxy BR from 13‐methoxy retinal and bacterioopsin is slower by a factor of 40 compared to 13‐ethyl BR. although both substituents are of similar size.

The position 13 of retinal was found to be most sensitive for regulation of the absorption maximum and the formation and stability of the all‐trans isomer, which is the active form for light‐induced proton translocation. The results suggest that an electronic interaction with a charged residue of the binding site exists around position 13 of retinal, which is disturbed when a methoxy group replaces the methyl or ethyl group at that position. This electronic interaction is essential for maintaining the active all‐trans configuration of retinal.
Amino‐acid sequence of ribonuclease T2 from Aspergillus oryzae
Authors: KAWATA Yasushi , SAKIYAMA Fumio , TAMAOKI Hidetsune
Published: March 2005

The amino acid sequence of ribonuclease T2 (RNase T2) from Aspergillus oryzae has been determined. This has been achieved by analyzing peptides obtained by digestions with Achromobacter lyticus protease I, Staphylococcus aureus V8 protease, and α‐chymotrypsin of two large cyanogen bromide peptides derived from the reduced and S‐carboxymethylated or S‐aminoethylated protein. Digestion with A. lyticus protease I was successfully used to degrade the N‐terminal half of the S‐aminoethylated protein at cysteine residues.

RNase T2 is a glycoprotein consisting of 239 amino acid residues with a relative molecular mass of 29155. The sugarcontent is 7.9% (by mass). Three glycosylation sites were determined at Asns 15, 76 and 239. Apparently RNase T2 has a very low degree of sequence similarity with RNase T1, but a considerable similarity is observed around the amino acid residues involved in substrate recognition and binding in RNase T1. These similar residues may be important for the catalytic activity of RNase T2.
Molecular characteristics and evidence for internalization of vasoactive‐intestinal‐peptide (VIP) receptors in the tumoral rat‐pancreatic acinar cell line AR 4‐2 J
Authors: SVOBODA Michal , DE NEEF Philippe , TASTENOY Michèle , CHRISTOPHE Jean
Published: March 2005

Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4‐2 J derived from rat pancreas [125I]Iodo‐VIP binding to cell membranes showed the following IC50 values for unlabeled peptides: VIP, 0.3 nM; peptide His‐IleNH2, 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [125I]iodo‐VIP binding to intact cells reached steady state after 5 min at 37°C, while the sequestration‐internalization of the [125I]iodo‐VIP ‐receptor complex (tested by cold acid washing) increased progressively, reaching 75% of total binding after 1 h. This phenomenon was blocked at 4°C. Further data with dexamethasone, tunicamycin, cycloheximide, low temperature, and/or phenylarsine oxide, suggested a half‐life of 2 days for VIP receptors and the necessity of N‐glycosylation for proper translocation.

For chemical [125I]iodo‐VIP cross‐linking bis[2‐(succinimidooxycarbonyloxy)ethyl]sulfone gave the best yield when compared with five other bifunctional reagents. In membranes, the main specifically cross‐linked peptide had MT 66000 under nonreducing conditions, and migrated with lower velocity (–5%) under reducing conditions. Cross‐linking was suppressed by VIP, peptide His‐IleNH2 and helodermin (competitively) and also by GTP. In intact cells, the Mr of [125I]iodo‐VIP‐cross‐linked peptides depended on the mode of cell solubilization. After direct solubilization, the major cross‐linked radioactivity migrated as a smear of Mr 130000–180000 but an MT‐66000 peptide was also detectable. In contrast, the solubilization of cross‐linked cells detached by mild trypsinisation gave mainly the MT‐66000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high‐MT complex and that mild cell treatment was sufficient to disrupt this complex.
Human liver cytosolic epoxide hydrolases
Authors: SCHLADT Ludwig , THOMAS Helmut , HARTMANN Renate , OESCH Franz
Published: March 2005

Human liver epoxide hydrolases were characterized by several criteria and a cytosolic cis‐stilbene oxide hydrolase (cEHcso) was purified to apparent homogeneity.

Styrene oxide and five phenylmethyloxiranes were tested as substrates for human liver epoxide hydrolases. With microsomes activity was highest with trans‐2‐methylstyrene oxide, followed by styrene 7, 8‐oxide, cis‐2‐With methylstyrene oxide, cis‐1,2‐dimethylstyrene oxide, trans‐1, 2‐dimethylstyrene oxide and 2, 2‐dimethylstyrene oxide. With cytosol the same order was obtained for the first three substrates, whereas activity with 2, 2‐dimethylstyrene oxide was higher than with cis‐1,2‐dimethylstyrene oxide and no hydrolysis occurred with trans‐1, 2‐dimethylstyrene oxide. Generally, activities were lower with cytosol than with microsomes.

The isoelectric point for both microsomal styrene 7, 8‐oxide and cis‐stilbene oxide hydrolyzing activity was 7.0, whereas cEHcso had an isoelectric point of 9.2 and cytosolic trans‐stilbene oxide hydrolase (cEHTSO) of 5.7.

The cytosolic epoxide hydrolases could be separated by anion‐exchange chromatography and gel filtration. The latter technique revealed a higher molecular mass for cEHcso than for cEHTSO.

Both cytosolic epoxide hydrolases showed higher activities at pH 7.4 than at pH 9.0, whereas the opposite was true for microsomal epoxide hydrolase. The effects of ethanol, methanol, tetrahydrofuran, acetonitrile, acetone and dimethylsulfoxide on microsomal epoxide hydrolase depended on the substrate tested, whereas both cytosolic enzymes were not at all, or only slightly, affected by these solvents.

Effects of different enzyme modulators on microsomal epoxide hydrolase also depended on the substrates used. Trichloropropene oxide and styrene 7, 8‐oxide strongly inhibited cEHcso whereas cEHTSO was moderately affected by these compounds.

Immunochemical investigations revealed a close relationship between cEHcso and rat liver microsomal, but not cytosolic, epoxide hydrolase. Interestingly, cEHTSO has no immunological relationship to rat microsomal, nor to rat cytosolic epoxide hydrolase. cEHTSO from human liver differed also from its counterpart in the rat in that it was only moderately affected by tetrahydrofuran, acetonitrile and trichloropropene oxide.

Five steps were necessary to purify cEHcso. The enzyme has a molecular mass (49 kDa) identical to that of rat liver microsomal epoxide hydrolase.
Synthesis and structural identification of four dihydroxy acids and 11, 12‐leukotriene C4 derived from 11, 12‐leukotriene A4
Authors: KITAMURA Shigeto , SHIMIZU Takao , MIKI Ichiro , IZUMI Takashi , KASAMA Takeshi , SATO Akira , SANO Hiroshi , SEYAMA Yousuke
Published: March 2005

Using a partially purified 12‐lipoxygenase from porcine leukocytes, (5Z,8Z,10E,14Z)‐12‐hydroperoxy‐5,8,10,14‐icosatetraenoic acid was synthesized from arachidonic acid with a yield of over 35%. The absolute configuration of C‐12 was determined as S by chiral‐phase column chromatography. It was chemically converted to at least three epoxides with the conjugated triene structure. Two were identified by proton NMR and mass spectrometry to be (5Z,7E, 9E,14Z)‐(11S, 12S)‐11,12‐oxido‐5,7,9,14‐icosatetraenoic acid (11,12‐leukotriene A4) and (5Z,7Z,9E,14Z)‐(11S,12S)‐11,12‐oxido‐5,7,9,14‐icosatetraenoic acid (7‐cis‐11,12‐leukotriene A4). 11,12‐Leukotriene A4 underwent acid hydrolysis to yield two diastereomers of (6E,8E,10E,14Z)‐(12S)‐5,12‐dihydroxy‐6,8,10,14‐icosatetraenoic acid and two isomers of (14Z)‐(12S)‐11,12‐dihydroxy‐5,7,9,14‐icosatetraenoic acid. Upon incubation with rat liver glutathione S‐transferase, 11,12‐leukotriene A4 was converted to 11,12‐leukotriene C4, a spasmogenic compound.
Structural study of long arm fragments of laminin
Authors: DEUTZMANN Rainer , HUBEP Johann , SCHMETZ Konrad A. , OBERBÄMER Ilse , HARTL Lydia
Published: March 2005

The outer segments of the long arm of laminin have recently been shown to mediate attachment of many cell types and to stimulate neurite outgrowth. For a structural characterization of this part of the molecule we prepared, by limited elastase digestion of laminin, fragments E3 and E8, previously identified as a globular heparin‐binding domain and as a 35‐nm‐long rod with a terminal globule, respectively. Fragment E3 is a domain adjacent to fragment E8. Both structures together comprise the complete terminal half of the long arm. Our data confirm current models, which predict that the C‐terminal segments from all three chains contribute to its structure. The B chains terminate at the end of the rod like domain, while the large terminal globule is formed by A‐chain structures only. In addition to fragment E3, two new fragments T1 and T2 obtained by tryptic cleavage of fragment E8 were characterized as substructures of the globular domain. Screening of a mouse cDNA library with synthetic oligonucleotides allowed isolation of an 1.8‐kb cDNA clone encoding 547 C‐terminal amino acids of the A chain and some 196 nucleotides of the 3′‐untranslated region including a single polyadenylation site. The clone contained portions of domain T2 and the complete heparin binding domain E3 which was thus identified as the most C‐terminal domain of the A chain. Sequence alignment indicated that the terminal globule is formed by homologous repeats of some 140 residues having no counterpart in the B chains.
An ATP‐dependent protease and ingensin, the multicatalytic proteinase, in K562 cells
Authors: TSUKAHARA Toshifumi , ISHIURA Shoichi , SUGITA Hideo
Published: March 2005

We investigated and characterized the ATP‐dependent protease in human erythroleukemia, K562 cells. The succinyl‐leucyl‐leucyl‐valyl‐tyrosine‐methylcoumarinamide hydrolytic activity in a K562 lysate at pH 9 rose more than 10‐fold with the addition of 1 mM ATP. The effect of ATP on the protease activity was dose‐dependent and inhibited by the addition of ADP. This activity was not inhibited by EDTA. L‐3‐carboxy‐trans‐2,3‐epoxypropionyl‐leucylamide‐(4‐guanidino)butane or leupeptin, but was strongly inhibited by chymostatin and diisopropyl‐fluorophosphate. The protease activity was eluted just after the void volume from a G3000SW HPLC column. The above results suggest that this protease is identical to the high‐molecular‐mass protease, ingensin, previously reported by us. The ATP‐dependent increase in the protease activity was due to prevention of the inactivation of the protease by ATP, and not to activation of the protease itself in the reaction mixture at 37°C. The depressed succinyl‐leucyl‐leucyl‐valyl‐tyrosine‐methylcoumarinamide hydrolytic activity in the ATP‐depleted lysate was restored to the same level by the detergent, SDS. Therefore, we conclude that the inactivation of ingensin occurring on preincubation is not irreversible.
Substrate‐specificity determinants for a membrane‐bound casein kinase of lactating mammary gland.
Authors: MEGGIO Flavio , BOULTON Antony P. , MARCHIORI Fernando , BORIN Gianfranco , LENNON David P. W. , CALDERAN Andrea , PINNA Lorenzo A.
Published: March 2005

A tissue‐specific casein kinase, purified from the Golgi‐enriched‐membrane fraction of guinea‐pig lactating mammary gland (GEF‐CK), readily phosphorylates the synthetic peptide Ser‐Glu5, a good substrate of casein kinase‐2, and several derivatives varying for the number and position of acidic residues on the C‐terminal side of serine, except those lacking an acidic side chain at position +2. The least acidic peptide, still significantly affected by GEF‐CK, is Ser‐Ala‐Glu‐Ala3 which is not a substrate for CK‐2. Conversely, the peptides Ser‐Ala2‐Glu‐Ala2, Ser‐Ala2‐Glu3, Ser‐Ala2‐Glu5 and Ser‐Glu‐Ala‐Glu3, all of which are more or less readily phosphorylated by CK‐2, are not appreciably affected by GEF‐CK. On the other hand the presence of additional glutamyl residues, besides the one in the second position, improves the affinity of the peptide substrate for GEF‐CK, as indicated by the Km values of Ser‐Glu5, Ser‐Glu2‐Ala3 and Ser‐Ala‐Glu‐Ala3 which are 80, 950 and 3950 μM respectively. It is concluded that although both CK‐2 and GEF‐CK require, for optimal activity, rather extended acidic clusters on the C‐terminal side of the target serine, the most critical residue in the case of GEF‐CK is not the one at position +3, which is required for CK‐2 catalyzed phosphorylation [Marin, O. et al. (1986) Eur. J. Biochem. 160, 239–244], but the one lying at position +2. Additional differences, concerning the site specificities of these enzymes, have been outlined using the threonyl derivative of Ser‐Glu5 and the peptide Arg‐Ser‐Glu3‐Val‐Glu. The former is still phosphorylated by CK‐2 but not to any appreciable extent by GEF‐CK, which apparently is strictly specific for seryl residues. On the contrary, the presence of an N‐terminal basic residue, which greatly reduces phosphorylation by CK‐2, is tolerated rather well by GEF‐CK. On the other hand a C‐terminal basic residue, interrupting the acidic cluster, compromises phosphorylation by GEF‐CK, as indicated by the extremely high Km value of Ser‐Glu3‐Lys‐Glu vs Ser‐Glu3‐Val‐Glu (13 000 and 170 μM, respectively).
The influence of water on protease‐catalyzed peptide synthesis in acetonitrile/water mixtures
Authors: RESLOW Mats , ADLERCREUTZ Patrick , MATTIASSON Bo
Published: March 2005

Protease‐catalyzed peptide synthesis in acetonitrile water mixtures, containing 0–90% water, was investigated. α‐Chymotrypsin, as well as thermolysin, were deposited on solid supports, prior to exposure to the reaction media. Peptide syntheses were performed using both a kinetically controlled process (chymotrypsin) and an equilibrium‐controlled synthesis (thermolysin). The activity of chymotrypsin decreased at low water contents. However, at low water contents (1–10%) hydrolytic side reactions were suppressed and high yields of dipeptides were obtained. Optimal water content for the thermolysin‐catalyzed reaction was 4–8%. The dipeptides produced were fully soluble in the reaction mixtures. High operational stability for α‐chymotrypsin was obtained during 216 h of reaction.
Tetraenoic and pentaenoic mycolic acids from Mycobacterium thamnopheos
Authors: DAFFÉ Mamadou , LANÉELLE Marie‐Antoinette , VALERO GUILLEN Pedro L.
Published: March 2005

On the basis of the analysis of mycolates, the type strain of Mycobacterium thamnopheos has been considered as a member of the genus Nocardia. In a comparative study conducted on mycobacterial species we found that M. thamnopheos synthesized two types of mycolate having the same mobilities on thin‐layer chromatography as those of mycobacteria, but different from nocardomycolates. Mass spectrometry analyzes showed that the major series of both types consisted of polyunsaturated mycolic acids, ranging from C72 to C78 with four or five double bonds. On pyrolytic mass spectrometry or gas chromatography, the least polar mycolates released mainly mono‐unsaturated C22 esters whereas the other type yielded saturated C20 and C22 esters. These results suggested that M. thamnopheos might be more related to the Aurantiaca taxon than to mycobacteria and Nocardia.

The permanganate‐periodate oxidation products of esters obtained by pyrolysis of the least polar mycolates showed that they contained docosen‐4‐oic and docosen‐6‐oic acids. Both types of mycolate esters yielded the same set of long‐chain meroaldehydes on pyrolysis. These meroaldehydes were significantly distinct from those of mycobacterial mycolates in the location of the double bonds.

After hydrogenation of the double bond located in the alkyl‐branched chain, the two types of mycolates had the same mobility on thin‐layer chromatography, indicating that the difference of migration was due to the additional double bond found in the least polar mycolates.

Based on stereochemical data, the relative configuration of both mycolates was found to be threo, like that established for all mycolates studied so far.
Genes coding for RNA polymerase β subunit in bacteria
Authors: LISITSYN Nicholay A. , MONASTYRSKAYA Galina S. , SVERDLOV Eugene D.
Published: March 2005

The nucleotide sequence of the rpoB gene of Salmonella typhimurium has been determined in this work. It was compared with known sequences of the gene from other sources and the conservative regions were detected. This allowed some interesting conclusions to be made about the distribution of the functional domains in bacterial RNA polymerase and about the three‐dimensional structure of its β subunit.
Investigation of protein structure by means of 19F‐NMR
Authors: ADRIAENSENS Peter , BOX Marie Elise , MARTENS Henri I. , ONKELINX Erik , PUT Jos , GELAN Jan
Published: March 2005

A 19F‐labeled derivative of hen egg‐white lysozyme, in which the six ɛ‐amino groups are trifluoroacetylated (LF,), was prepared by reaction of lysozyme with S‐ethyltrifluorothioacetate. The reaction mixture was fractionated by cation‐exchange chromatography at pH 7.3.

A comparison of the circular dichroic spectra and the activity towards Micrococcus lysodeikticus of both LF6 and native lysozyme reveals that the labeling causes no major conformational changes of the polypeptide backbone.

Assignment of the six resonances present in the 19F‐NMR spectrum of LF6 was accomplished by using a variety of techniques: specific chemical modifications, the effect of the inhibitor (GlcNAc)3, 19F‐shift/pH information and relaxation parameters.
Tyrosyl‐tRNA synthetase from baker's yeast
Authors: FREIST Wolfgang , STERNBACH Hans
Published: March 2005

The order of substrate addition to tyrosyl‐tRNA synthetase from baker's yeast was investigated by bisubstrate kinetics, product inhibition and inhibition by dead‐end inhibitors. The kinetic patterns are consistent with a random bi‐uni uni‐bi ping‐pong mechanism.

Substrate specificity with regard to ATP analogs shows that the hydroxyl groups of the ribose moiety and the amino group in position 6 of the base are essential for recognition of ATP as substrate.

Specificity with regard to amino acids is characterized by discrimination factors D which are calculated from kcat and Km values obtained in aminoacylation of tRNATyr‐C‐C‐A. The lowest values are observed for Cys. Phe. Trp (D= 28000 – 40000), showing that, at the same amino acid concentrations, tyrosine is 28000 – 40000 times more often attached to tRNATyr‐C‐C‐A than the noncognate amino acids. With Gly, Ala and Ser no misacylation could be detected (D > 500000); D values of the other amino acids are in the range of 100000—500000.

Lower specificity is observed in aminoacylation of the modified substrate tRNATyr‐C‐C‐A(3'NH2) (D1= 500—55000). From kinetic constants and AMP‐formation stoichiometry observed in aminoacylation of this tRNA species, as well as in acylating tRNATyr‐C‐C‐A hydrolytic proof‐reading factors could be calculated for a pretransfer (II1) and a post‐transfer (II2) proof‐reading step. The observed values of II1= 12 – 280 show that pretransfer proof‐reading is the main correction step whereas post‐transfer proof‐reading is marginal for most amino acids (II2= 1–2).

Initial discrimination factors caused by differences in Gibbs free energies of binding between tyrosine and noncognate amino acids are calculated from discrimination and proof‐reading factors. Assuming a two‐step binding process, two factors (I1 and I2) are determined which can be related to hydrophobic interaction forces. The tyrosine side chain is bound by hydrophobic forces and hydrogen bonds formed by its hydroxyl group. A hypothetical model of the amino acid binding site is discussed and compared with results of X‐ray analysis of the enzyme from Bacillus stearothermophilus.
Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I‐69 Rd−/b+
Authors: HELANDER Ilkka M. , LINDNER Buko , BRADE Helmut , ALTMANN Klaus , LINDBERG Alf A. , RIETSCHEL Ernst Th. , ZÄHRINGER Ulrich
Published: March 2005

The chemical structure of the lipopolysaccharide of a deep‐rough mutant (strain I‐69 Rd−/b+) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a β‐(1′,6)‐linked D‐glucosamine disaccharide with phosphate groups at C‐1 of the reducing D‐glucosamine and at C‐4′ of the nonreducing one. Four molecules of (R)‐3‐hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2, 2′ and 3, 3′, respectively). Laser‐desorption mass spectrometry showed that both 3‐hydroxytetradecanoic acids linked to the non‐reducing glucosamine carry tetradecanoic acid at their 3‐hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A.

The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583–587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas‐liquid chromatography/mass spectrometry) also revealed dOclA 5‐phosphate. Since a cyclic 4,5‐phosphate could be ruled out by 31P‐NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4‐ and 5‐phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C‐6′ of the nonreducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by 1H‐NMR and 13C‐NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure:

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Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins
Authors: GROSS Hans Jürgen , BROSSMER Reinhard
Published: March 2005

Aiming at the introduction of a fluorescent sialic acid into glycoconjugates, 5‐acetamido‐9‐(3‐fluoresceinylthioureido)‐3,5,9‐trideoxy‐2‐nonulosonic acid (9‐fluoresceinyl‐NeuAc) was synthesized which has an intact carbon chain.

a) Despite the space‐filling substituent at C‐9 the fluorescent NeuAc analogue was activated to the corresponding CMP‐glycoside by CMP sialic acid synthase from bovine brain. Whereas the km value of the synthase was little affected by the modification (Km= 2.1 mM, for NeuAc Km= 1.4 mM), the V value decreased to 7.5%.

b)CMP‐9‐fluoresceinyl‐NeuAc was synthesized on a preparative scale (17% overall yield), and characterized by analytical HPLC, absorption and fluorescence spectra.

c) 9‐Fluoresceinyl‐NeuAc was transferred onto asialo‐α1‐acid glycoprotein by both Galβ1, 4GlcNAc α2, 6sialyltransferase and Galβ1,4(3)GlcNAc α2,3sialyltransferase (rat liver), and onto antifreeze glycoprotein by GalNAc α2,6‐sialyltransferase (porcine submaxillary glands). Using analytical HPLC, transfer was confirmed after release of the fluorescent sialic acid by Vibrio cholerae sialidase.

d) Initital rate studies indicated a low Km value of Galβ‐1,4GlcNAc α2,6sialyltransferase, and GalNAc α2,6sialyltransferase (specific for O‐linked oligosaccharide chains) for CMP‐9‐fluoresceinyl‐NeuAc.
Inhibitors of liver lysosomal acid phospholipase A1
Authors: KUNZE Hans , BOHN Ernst , LÖFFLER Bernd‐Michael
Published: March 2005

Lysosomal acid phospholipase A1, as well as other lysosomal enzymes, may be released under patho‐physiological conditions into extralysosomal compartments. As shown here, several unspecific mechanisms exist which inhibit the hydrolysis of membrane diacylphospholipids by lysosomal acid phospholipase A1 and hence prevent an uncontrolled membrane destruction. These findings were obtained by employing partially purified rat liver lysosomal acid phospholipase A1 and sonicated radioactively labeled phosphatidylethanolamine or phosphatidylcholine as substrate. The inhibitory principles found include (1) pH, (2) inorganic cations, and (3) various proteins. Inorganic cations and proteins, however, inhibited lysosomal acid phospholipase A1 activity only below pH 6.0, and inhibition never exceeded 96%. Of the inorganic cations studied, the divalent species, as compared to the monovalent one, impaired lysosomal acid phospholipase A1 activity at significantly lower concentrations. Virtually all of the intracellular and extracellular proteins studied inhibited the enzyme activity, but the inhibitory potencies of the different proteins varied considerably. In general, basic and hydrophobic proteins were the most potent inhibitors, whereas glycoproteins appeared to be less inhibitory. The degree of inhibition of the enzyme activity in both proteins and inorganic cations depended on the substrate concentration and not on that of the enzyme. Binding studies provided evidence for inhibitor‐substrate and against inhibitorenzyme interactions.
Temperature‐dependent conformational transition in the head‐rod junctional region of the myosin molecule
Authors: RĘDOWICZ Maria J. , STRZELECKA‐GOLASZEWSKA Hanna
Published: March 2005

The effects of temperature, Mg2+, ATP, and actin on the conformation of the neck region of the myosin head were studied by limited proteolysis of heavy meromyosin (HMM) and subfragment 1 (S1) preparations obtained by papain digestion of myosin in the presence of Mg2+ (Mg‐S1) or EDTA (EDTA‐S1). The preparations were fluorescently labelled at the SH1 thiol group to enable identification of the COOH‐terminal fragments of the head portion of the heavy chain where this group is located.

The results indicate that the head‐rod junctional region of the myosin heavy chain contains at least three different sites readily susceptible to trypsin at 25°C if the light chain LC2 or its LC′2 fragment are absent. The susceptibility of one of these sites dramatically decreases when the temperature is lowered to 0°C, indicating a temperature‐dependent conformational transition in the head‐rod junction. With the method used, this transition is detectable only in LC2/LC′2‐deficient preparations since all three sites are protected, although to different extents, by LC2 and its LC′2 derivative. It is, however, most probable that the effect of the light chain is confined to steric hindrance of trypsin access and that the temperature‐dependent structural transition in the head‐rod junction can occur in the presence of intact LC2 as well and may contribute to the temperature sensitivity of force generation in muscle.
A 15000‐Mr protein proteolytically derived from vitellogenin within oocyte of Perinereis cultrifera (polychaete annelid)
Authors: BAERT Jean‐Luc
Published: March 2005

It has been shown previously in our laboratory that, in Perinereis cultrifera, the four mature vitellin subunits (Mr 98000, 22000, 20000, 16000) are proteolytically derived within the oocyte from a single extraoocytic precursor, vitellogenin, with an apparent Mr (176000) higher by 20000 than the sum of the Mr of the four end products. In this report, it is shown that a 15000‐Mr protein, designated as P15, not only accumulates in maturing oocytes but also originates from outside these cells similarly to vitellin. Moreover in vivo labelling experiments indicate that the appearance of P15 occurs after vitellogenin enters the oocyte, concurrently with the appearance of the lower‐Mr fragments characteristic of vitellin. From these data, it is concluded that P15 most likely represents a vitellogenin‐derived protein which is generated within the oocytes during the transformation of vitellogenin into vitellin. This conclusion is further supported by the additional finding that P15 immunologically cross‐reacts with vitellogenin but not with mature vitellin.

P15 has been purified to homogeneity from the soluble protein fraction of submature oocytes and partially characterized. The 15000‐Mr protein exists in a monomeric form with a pI of about 7.7. Unlike vitellin, P15 does not contain significant amounts of lipid or carbohydrate and has a low absorbance at 280 nm. The amino acid composition of the purified protein is also presented.
Fructose 2,6‐bisphosphate and the climacteric in bananas
Authors: BALL Kathryn L. , AP REES Tom
Published: March 2005

This work was done to test the view that there is a marked rise in the content of fructose 2,6‐bisphosphate during the climateric of the fruit of banana (Musa cavendishii Lamb ex. Paxton). Bananas were ripened in the dark in a continuous stream of air in the absence of exogenous ethylene. CO2 production and the contents of fructose 2,6‐bisphosphate and sucrose were monitored over a 15‐day period. A range of extraction procedures for fructose 2,6‐bisphosphate were compared. Recovery of fructose 2,6‐bisphosphate added to samples of unripe fruit varied from poor to unmeasurable. Recoveries from samples of ripe fruit were high. It is argued that this differential recovery of fructose 2,6‐bisphosphate undermines claims that the amount of this compound increases at the climacteric. When recoveries are taken into account, our data suggest that there is no major change in fructose 2,6‐bisphosphate content during the onset of the climacteric in bananas.
Platelet‐activating‐factor‐induced serum‐albumin release from rabbit platelets in the absence of calcium
Authors: BETTE‐BOBILLO Pascale , BIENVENUE Aain
Published: March 2005

Using a radioimmunoassay for rabbit‐serum albumin, platelet‐activating‐factor‐induced serum‐albumin release by platelets was monitored under non‐aggregating conditions. The four main results from this study are as follows. The EC50 of the release was of the same order of magnitude as the aggregation EC50 in the presence of calcium. The release took place within 2 min and was inhibited by BN 52021, which is a very specific inhibitor of the platelet‐activating‐factor‐aggregating effect. Serum‐albumin release was much greater than serotonin release.