bluuebunny/crossref_metadata_2025_split huggingface dataset:
Collagen‐binding domain of human plasma fibronectin contains a latent type‐IV collagenase
Authors: LAMBERT VIDMAR Smilja , LOTTSPEICH Friedrich , EMOD Istvan , IMHOFF Jean‐Marie , KEIL‐DLOUHA Vera 
Published: March 2005

Cleavage of the 45‐kDa gelatin‐binding fragment of human plasma fibronectin with fibronectinase resulted in the activation of two forms of metalloproteinase with different substrate specificities. The 40‐kDa FN‐type‐IV collagenase A degrades heat‐denatured type‐I collagen, laminin and also native collagen type IV. The 27‐kDa FN‐type‐IV collagenase B degrades native collagen type IV, but it does not cleave laminin and only poorly degrades gelatin. Both enzymes begin with the same N‐terminal sequence VYQPQPH‐ (residues 262–268 of fibronectin) but, contrary to the FN‐type‐IV collagenase A, the FN‐type‐IV collagenase B has lost the C‐terminal region of type I repeats, where the major gelatin‐binding determinants of fibronectin are located. The FN‐type‐IV collagenases A and B are sequentially similar to the middle domain (domain II) of collagenase type IV, secreted by H‐ras‐transformed human bronchial epithelial cells.Substrate and inhibition specificity of FN‐type‐IV collagenase A and B are different from those of FN‐gelatinase and FN‐laminase, isolated previously from the central and C‐terminal fibronectin domains, respectively. The substrate specificity of both enzymes, characterized in this study, is also different from that of already known matrix‐degrading metalloproteinases.
N‐Myristoyl‐transferase activity in cancer cells
Authors: BOUTIN Jean Albert , CLARENC Jean‐Pierre , FERRY Gilles , ERNOULD Anne‐Pascale , REMOND Georges , VINCENT Michel , ATASSI Ghanem 
Published: March 2005

The activity catalyzed by N‐myristoyl tranferase (NMT) is described for the first time in microsome‐rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accomodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N‐terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively).As for some other types of membrane‐bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome‐rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificy.Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome‐associated activity. None of them show any significant potency in inhibiting this activity.A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (IC50) of 0.2 mM.
Specificity determinants for tyrosine protein kinases
Authors: DONELLA‐DEANA Arianna , STONE Stuart R. , PINNA Lorenzo A. 
Published: March 2005

Hirudin, the powerful anticoagulant agent of leech (Hirudo medicinalis) saliva, was readily phosphorylated by two spleen tyrosine protein kinases (TPK‐IIB and TPK‐III) at Tyr63 withKm values of 238 μM and 74 μM, respectively. The synthetic tridecapeptide DGDFEEIPEEYLQ, corresponding to the hirudin 53–65 C‐terminal fragment, was phosphorylated even more efficiently than hirudin itself. Four hirudin mutants, in which one or more of the glutamic acids at positions 57, 58, 61 and 62 have been replaced by glutamines, were poorer substrates than hirudin. The mutant in which all four glutamates were substituted, [Gln57, 58, 61, 62]hirudin, was virtually not phosphorylated by either TPK‐IIB. Substitution of Glu57 and Glu58 was less deleterious than substitution of the two glutamic acids adjacent to Tyr63: [Gln61,62]hirudin exhibited a 20‐fold lower phosphorylation efficiency with TPK‐IIB. With TPK‐III, however, theKm value of[Gln61,62]hirudin was slightly lower, while theVmax decreased sixfold. The substitution of Glu62 alone was also more detrimental with TPK‐IIB than with TPK‐III. The behaviour of a third spleen TPK, namedlyn TPK‐I and belonging to thesrc family, was markedly different in that it did not phosphorylate hirudin but exhibited significant activity towards [Gln57, 58, 61, 62]hirudin.Taken together, these data confirm and extend with a protein substrate the results obtained with short model peptides which indicated the stringent substrate requirements of TPK‐IIB (and of TPK‐III to a lesser extent) for N‐terminal acidic residues. In contrast, such residues are deleterious withlyn TPK‐I. These observations also support the concept that tyrosine protein kinases recognize specificity determinants situated in the vicinity of the target residue rather than requiring higher‐order structural features.
Mechanism of NADPH oxidation catalyzed by horse‐radish peroxidase and 2,4‐diacetyl‐[2H]heme‐substituted horse‐radish peroxidase
Authors: DE SANDRO Virginie , DUPUY Corinne , KANIEWSKI Jacques , OHAYON Renée , DÈME Danielle , VIRION Alain , POMMIER Jacques 
Published: March 2005

The mechanism of NADPH oxidation catalyzed by horse‐radish peroxidase (HRP) and 2,4‐diacetyl‐[2H]heme‐substituted horse‐radish peroxidase (DHRP) was studied. The roles of the different H2O2/peroxidase compounds were examined by spectral studies. The oxidized NADPH species were identified using the superoxide dismutase effect and by measuring the stoichiometry between NADPH oxidized and H2O2 used.In the presence of a mediating molecule, like scopoletin, both enzymes acted via a similar mechanism, producing only NADP°, which in turn reacted with O2 producing O2. Consequently H2O2 was completely regenerated in the presence of superoxide dismutase and partially regenerated in its absence.In the absence of a mediating molecule, the H2O2 complex of both enzymes (compound I) catalysed NADPH oxidation by single‐electron transfer, producing NADP°; compound II of these enzymes catalyzed NADPH oxidation more slowly by a direct two‐electron transfer, producing NADP+.There were difference between HRP and DHRP. HRP compound II was produced by the oxidation of 1 mol NADPH/mole compound I, while DHRP compound II was formed by the spontaneous conversion of compound I to compound II. The NADPH oxidation catalyzed by DHRP compound I did not lead to the formation of compound II. When H2O2 was produced slowly by the glucose/ glucose‐oxidase system, compound II was never formed and a pure O2 adduct of DHRP (compound III) accumulated.
Structure/activity relationship of eel calcitonin
Authors: INOUE Akira , SHIKANO Mayumi , KOMATSU Yasuhiko , OBATA Junko , OCHIAI Junko , NISHIDE Hiroko , ITO Noriko , NAGAO Hiromasa , KONDO Kiyoshi , TUNEMOTO Daiei , HEMMI Hiromichi , NUMAO Naganori 
Published: March 2005

A series of analogs of eel calcitonin (eCT) was synthesized according to a newly devised scheme, ‘the insertion‐inactivation method’, to clarify the structure/activity relationship of a given peptide. This method consists of two steps: the deletion of a residue of the peptide is first chosen and then a series of analogs with the residue reinserted into serial positions is synthesized and biological activities are assessed in each step.An analog lacking Lys18 (dK), selected as a deleted analog for the first step, showed marked loss of activities determined by inhibition of 125I‐eCT binding, growth inhibition, and cAMP production in a porcine kidney cell line LLC‐PK1. Activities of a set of 20 analogs with the reinserted lysine residue at serial positions from 12 to 32 (K12–K32) were then evaluated. The results showed the following three patterns of the expression of activities according to the position of the reinsertion: (a) analogs K12–K16 (positions 12–16) and K25 (position 25) showed lower activities than eCT in all assays; (b) K17–K24 (positions 17–24) showed slightly lower activities than eCT in the receptor binding and the growth inhibition and similar level in cAMP production; (c) K26–K32 (positions 26–32) showed considerably lower activities in the former two assays and slightly lower activity in cAMP production. Further, analogs considerably less active than eCT showed unchanged α‐helix contents and destroyed amphiphilicity by the insertion of a lysine residue, indicating that amphiphilicity is one of important factors for expressing the activity.The results obtained here lead to a conclusion on the significance of each region of eCT molecule as follows: (a) the presence of Lys18 is necessary for the complete expression of biological activity; (b) the length of amphiphilic α‐helix to be required for the activity is at most 10 residues ranging from position 8 to position 17; (c) the receptor binding region is located within 9 residues ranging from position 24 to position 32.
Multiple mRNA species code for two non‐allelic forms of ovine αs2‐casein
Authors: BOISNARD Monique , HUE Dominique , BOUNIOL Christine , MERCIER Jean‐Claude , GAYE Pierre 
Published: March 2005

The two non‐allelic forms of αs2‐casein, occurring in ovine milk, differ by an internal deletion of nine amino acid residues, at positions 34 and 42 in the mature chain. Sequencing of several αs2‐casein cDNA, isolated from the mammary cDNA library of a single lactating ewe, showed three new types which differed from that previously studied. In addition to the expected deletion of codons + 34 to + 42 affecting 30–40% of mRNA, another structural difference involving an internal stretch of 44 nucleotides in the 5′untranslated region, was found. S1‐nuclease protection assays confirmed the existence of several types of the relevant mRNA and sequencing of in‐vitro‐amplified genomic DNA demonstrated the presence of the 44‐nucleotide stretch in the αs2‐casein transcriptional unit, thus ruling out the possibility of a cloning artefact.The different αs2‐casein mRNA, four in terms of deletion and two in terms of nucleotide substitutions for a given ewe, can be readily explained by partial exon skipping and allelic differences, respectively. This assumption is well supported by the following observations: 5′ and 3′ ends of both deleted DNA fragments are similar to those of exons; sequences neighbouring the 44‐nucleotide stretch of the genomic DNA perfectly match consensus sequences described for 3′ and 5′ ends of introns; the rather simple patterns observed on Southern blots of different enzymatic digests of genomic DNA strongly suggest the occurrence of only 1 copy αs2‐casein gene/haploid genome. During the course of evolution, the αs2‐casein‐encoding gene has undergone many mutations and some of them might have occurred in regions corresponding to consensus splicing regions of the pre‐mRNA. Thus, complete skipping of some exons might be responsible for the shorter sizes of rat and mouse αs2‐casein mRNA. If so, the overall organization of the αs2‐casein gene in the different species might be more similar than expected from structural comparisons of the cognate mRNA or caseins.
1H‐NMR conformational analysis of a high‐affinity antigenic 11‐residue peptide from the tryptophan synthase β2 subunit
Authors: DELEPIERRE Muriel , LARVOR Marie‐Pierre , BALEUX Françoise , GOLDBERG Michel E. 
Published: March 2005

Two synthetic peptides from the β2 subunit of tryptophan synthase have been studied by 1H‐NMR spectroscopy at 300 MHz. One peptide, His‐Gly‐Arg‐Val‐Gly‐Ile‐Tyr‐Phe‐Gly‐Met‐Lys (peptide 11; Ile, isoleucine) is antigenic and binds with a high affinity to a monoclonal antibody that recognizes the native β2 subunit. The second peptide, His‐Gly‐Arg‐Val‐Gly‐Ile‐Tyr‐Phe (peptide 8) reacts very weakly with the antibody. The 1H‐NMR spectra of the two peptides have been assigned from two‐dimensional techniques in H2O, 2H2O and (2H6) dimethyl sulfoxide [(2H6)Me2SO]. The structure has been evaluated through analysis of nuclear Overhauser effects, coupling constants, amide‐proton exchange rates and their temperature coefficients, and chemical shifts.In aqueous solvent, the C‐terminal part of peptide 11 presents some structure centered around residues Phe‐Gly‐Met. The relationship between the structure found in peptide 11 and its antigenic nature is discussed.
Spectroscopic characterization of true enzyme‐substrate intermediates of aspartate aminotransferase trapped at subzero temperatures
Authors: STERK Michael , GEHRING Heinz 
Published: March 2005

Absorption and circular dichroism spectra of stable enzyme‐substrate intermediates of aspartate aminotransferase were recorded at subzero temperatures (down to ‐65°C) in the cryosolvent water/methanol. The intermediates were formed either between the pyridoxal from of the enzyme and its amino acid substrates, or between the pyridoxamine form and its oxo acid substrates. K′d values determind by spectroscopic titration were very close to the Km values reported for the different substrates. The adsorption complex of the pyridoxal form was probably obtained on addition of cysteine sulfinate. This complex is characterized by an increased absorption at 430nm together with a positive Cotton effect, as also observed in the case of the complex with the competitive inhibitor maleate indicating protonation of the internal aldimine. Addition of the substrates aspartate or glutamate to the pyridoxal form seemed to result in the direct accumulation of the external aldimine which showed a slight decrease in both the absorbance and the Cotton effect at 360nm. Additionally, a bathochromic shift of 5nm was observed in the case of glutamate. At 430nm, only a minor increase in absorbance, but not in circular dichroism, was observed with aspartate, and no changes were found with glutamate and the substrate analog 2‐methylaspartate, indicating a deprotonated external aldimine. Presumably, the ketimine intermediate was obtained on addition of the oxo acids 2‐oxoglutarate or oxalacetate to the pyridoxamine from. The intemediate showed a slight bathochromic shift (2nm) of the absorption band and decreased circular dichroism. On formation of the ketimine, a tyrosine residue, probably active‐site Tyr225, becomes partly ionized.The finding that the external aldimine can probably be accumulated in the conversion of the pyridoxal to the pyridoxamine from with the natural substrates would confirm the proton abstraction at Cα to be the rate‐limiting step in the tautomerization, although with cysteine sulfinate, the formation of the external aldimine might contribute to the rate limitation. Accumulation of the ketimine in the reverse direction would indicate that the proton abstraction at C4′ is rate‐limiting in this half‐reaction. The results demonstrate the feasibility of further structural investigations of true enzyme‐substrate intermediates.
Characterization, stability and refolding of recombinant hirudin
Authors: OTTO Angelika , SECKLER Robert 
Published: March 2005

A recombinant variant of hirudin, the blood‐clotting inhibitor of the leech Hirudo medicinalis, has been characterized employing spectroscopic and hydrodynamic techniques. Conditions have been defined for efficient reconstitution of the native, disulfide‐bonded inhibitor from completely unfolded, reduced polypeptide chains.The spectral properties of the native inhibitor are consistent with previous results on the solution structure of hirudin. Extremely low circular dichroism in the far ultraviolet () indicates a very low content of regular secondary structure. Although both tyrosine residues of the recombinant inhibitor titrate around pH 10.6, typical for solvent‐exposed tyrosines, fluorescence emission and near‐ultraviolet circular dichroism suggest that at least one of the tyrosines is partially shielded from solvent quench, and immobilized in an asymmetric environment. Reversible thermal unfolding of hirudin around 65° C is indicated by the disappearance of its dichroic absorption in the near ultraviolet and by a fourfold increase in ellipticity at 225 nm. The transition can be approximated by a two‐state model with a transition enthalpy of Hvan't Hoff= 159 kJ/mol and a transition entropy of 464 J · mol−1· K−1. Reduced hirudin at room temperature is largely unfolded and inactive as an inhibitor of thrombin assayed with a low‐molecular‐mass substrate. Refolding and reoxidation are observed at alkaline pH in the presence of a mixture of glutathione and glutathione disulfide. Spectroscopy, thrombin inhibition, and reversed‐phase HPLC indicate reconstitution yields close to 100% and that the reconstituted inhibitor is identical to the native starting material.
Expression of rat heme oxygenase in Escherichia coli as a catalytically active, full‐length form that binds to bacterial membranes
Authors: ISHIKAWA Kazunobu , SATO Michihiko , YOSHIDA Tadashi 
Published: March 2005

A plasmid, pKK‐RHO, was constructed by incorporating the coding sequence of a cDNA for rat heme oxygenase into the expression vector pKK233‐2. Escherichia coli strain XL1‐blue transformed with pKK‐RHO produced a catalytically active, full‐length heme oxygenase. The 32‐kDa native enzyme expressed, was localized in the bacterial membranes, possibly due to the spontaneous membrane‐binding properties of a hydrophobic segment in its C‐terminal region. During cultivation, a few degraded forms of heme oxygenase that had lost their membrane‐associative properties appeared. Probably, some bacterial proteases cut the native heme oxygenase at sites near its C‐terminus and so release hydrophilic peptides of heme oxygenase from the membranes. A 30‐kDa polypeptide, one of the degraded forms of heme oxygenase, retained ability to accept electrons from NADPH‐cytochrome P450 reductase and also activity for catalyzing breakdown of heme to biliverdin.The cultured cells were pale green. From them we extracted green pigment(s), of which the absorption spectrum closely resembled that of biliverdin, suggesting that a large amount of the endogenous heme of E. coli was actually degraded to biliverdin by the expressed heme oxygenase.
Biochemical and biophysical investigations of the ferrocene‐iron‐loaded rat
Authors: WARD Roberta J. , FLORENCE Anne L. , BALDWIN Dianne , ABIAKA Clifford , ROLAND Francine , RAMSEY Michael H. , DICKSON Dominic P. E. , PETERS Timothy J. , CRICHTON Robert R. 
Published: March 2005

Male Wistar rats fed with ferrocene had high hepatic iron loading (7.24 ± 1.97 mg Fe/g tissue) after 6 weeks, principally located in lysosomes, which was comparable to the levels and distribution determined in human haemochromatosis.The two iron‐storage proteins, ferritin and haemosiderin were isolated from the livers of the ferrocene‐loaded rats and their iron cores were investigated by Mössbauer spectroscopy and inductively coupled plasma‐emission spectrometry. Ferrihydrite was the predominant form of iron present in both ferritin and haemosiderin, while haemosiderin contained higher amounts of phosphorus, magnesium, calcium and barium, then either normal or ferrocene‐loaded ferritin.Free‐radical‐mediated damage in the iron‐loaded livers was inferred by the significant depletion of α‐tocopherol in both the livers and subcellular hepatic lysosomal fraction, which inversely correlated with the increasing iron content (r=–0.61; P < 0.05) and was associated with increased fragility of the lysosomal membranes.
Expression of CYP2D6 in developing human liver
Authors: TRELUYER Jean‐Marc , JACQZ‐AIGRAIN Evelyne , ALVAREZ Fernando , CRESTEIL Thierry 
Published: March 2005

The CYP2D6 protein is a polymorphic isoenzyme involved in the biotransformation of several drugs including the probe drug dextromethorphan. The rise in the protein concentration, immunochemically determined with a specific antibody, was shown to occur within the first week following birth, whatever the gestational age at birth.In fetuses, the concentration of hepatic CYP2D6 protein was very low or undetectable in 70% of samples tested. In the remaining 30%, its concentration was comparable to that of newborns aged 1–7 days. This early rise was associated with spontaneous abortion in 70% of positive samples, whereas in fetuses with an intermediate CYP2D6 protein concentration, 80% were from induced abortions.The rise in CYP2D6 protein was associated with the developmental onset of dextromethorphan O‐demethylation, but not N‐demethylation, even if activity was lower in fetal than in neonatal and in adult liver microsomes.Lastly, the CYP2D6 RNA is detectable earlier than the protein and exhibits a peak of hepatic accumulation in newborns, before declining in adulthood. A positive correlation between RNA accumulation and protein concentration can be demonstrated only in the adult. This suggests that regulation is primarily at the transcriptional level, but cannot rule out the participation of post‐transcriptional events in the regulation process throughout ontogenesis.
Mollusc‐specific toxins from the venom of Conus textile neovicarius
Authors: FAINZILBER Michael , GORDON Dalia , HASSON Arik , SPIRA Micha E. , ZLOTKIN Eliahu 
Published: March 2005

Three peptide toxins exhibiting strong paralytic activity to molluscs, but with no paralytic effects on arthropods or vertebrates, were purified from the venom of the molluscivorous snail Conus textile neovicarius from the Red Sea. The amino acid sequences of these mollusc specific toxins are: TxIA, WCKQSGEMCNLLDQNCCDGYCIVLVCT (identical to the so called ‘King Kong peptide’); TxIB, WCKQSGEMCNVLDQNCCDGYCIVFVCT; TxIIA, WGGYSTYCγVDSγCCSDNCVRSYCT (γ=γ‐carboxyglutamate).There is a similarity of the Cys framework of these toxins to that of the ω‐conotoxins; however, their net negative charges, high content of hydrophobic residues and uneven number of Cys residues in TxIIA, are highly unusual for conotoxins. When assayed on isolated cultured Aplysia neurons, all three toxins induced membrane depolarization and spontaneous repetitive firing. The TxI toxins also induce a marked prolongation of the action potential duration, which is sodium dependent. These effects differ significantly from the blocking activities of piscivorous venom conotoxins. These mollusc specific conotoxins may therefore serve as new and selective probes for ion‐channel functions in molluscan neuronal systems.
αB‐crystallin exists as an independent protein in the heart and in the lens
Authors: BHAT Suraj P. , HORWITZ Joseph , SRINIVASAN Anupama , DING Linlin 
Published: March 2005

αB‐crystallin, a polypeptide of molecular mass 22 kDa, is considered to be one of two subunits (αA and αB) of the multimeric lens‐specific protein, α‐crystallin. Recent demonstrations of the extralenticular presence of αB‐crystallin have suggested that outside of the lens, this polypeptide may have functions independent of αA. Within the lens however, as part of the protein α‐crystallin, its function is assumed to be structural. In an effort to investigate the functional status of αB‐crystallin in the lens, we have characterized this polypeptide in the rat heart and the human lens.Unequivocal identity of αB‐crystallin in the rat heart and the rat lens was established by the sequence analyses of the respective cDNA clones. Size exclusion chromatography (FPLC) and immunoblotting showed that in the rat heart, αB‐crystallin exists as an aggregate of 300–400 kDa average molecular mass, similar to that of purified αB‐crystallin isolated from bovine lens. Interestingly, analysis of the human lens proteins by immunoblotting showed that, with age, unlike αA‐crystallin, the αB subunit remains detectable in the soluble fractions derived from normal lenses as old as 82 years. Importantly, the average molecular mass of the αB subunit in the soluble fractions prepared from 60–80‐year‐old human lens nuclei was also found to be 300–400 kDa. These data lead to the conclusion that αB‐crystallin may exist as an independent protein not only in non‐lens tissues (e.g. heart) but in the lens as well.