bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear Substrates of Protein Kinase C
Authors: BECKMANN Roland , BUCHNER Klaus , JUNGBLU Peter R. , ECKERSKORN Christoph , WEISE Christoph , HILBERT Ralf , HUCHO Ferdinand
Published: March 2005

Starting from the finding that, for neuronal cells, the nuclear‐membrane‐associated protein kinase C (PKC) is the so‐called ‘membrane inserted’, constitutively active form, we attempted to identify substrates of this nuclear PKC. For this purpose, nuclear membranes and other subcellular fractions were prepared from bovine brain, and in‐vitro phosphorylation was performed. Several nuclear membrane proteins were found, the phosphorylation of which was inhibited by specific PKC inhibitors and effectively catalyzed by added PKC. Combining the methods of two‐dimensional gel electrophoresis, in‐situ digestion, reverse‐phase HPLC and microsequencing, two of these nuclear PKC substrates were identified; the known PKC substrate Lamin B2, which serves as a control of the approach and the nucleolar protein B23. Our data suggest, that, for B23, Ser225 is a site of phosphorylation by PKC.
The use of multiple‐pin peptide synthesis in an analysis of the continuous epitopes recognised by various anti‐(recombinant bovine growth hormone) sera
Authors: BEATTIE James , FAWCETT Hilary A. C. , FLINT David J.
Published: March 2005

A recently developed technology called epitope scanning permits the rapid and accurate delineation of continuous stretches of amino acids in a protein which constitute the sequential epitopes recognised by an antiserum raised to that protein. In the present report, we describe the use of this technique to identify the epitopes in the recombinant bovine growth‐hormone (rbGH) molecule recognised by three polyclonal guinea‐pig antisera and two polyclonal rabbit antisera. The results obtained show that, for guinea‐pig antisera, 3 or 4, very‐well‐defined major continous epitopes are present. As would be expected given the intrinsic genetic factors (major histocompatibility restriction, antigen processing and presentation) controlling the immune response in individual animals, subtle differences are evident in the precise location and relative reactivities of these epitopes in different guinea‐pig antisera. Nevertheless, there is a large degree of overlap in these epitopes, such that immunodominant regions of the antigen can be clearly delineated. In a structural sense, these epitopes share a common motif in that they are sited in areas of the protein antigen with little secondary structure (loop/coil), although there is some contribution by neighbouring α‐helices. For the two rabbit antisera, the response tends to be rather more heterogeneous, with recognition of more peptides and less clearly defined epitopes than was the case with the guinea‐pig antiserum.

Comparison of the four guinea‐pig epitopes, identified by our experimental methods with computer predictions for this molecule (Jameson‐Wolf antigenic index), indicate that two are strongly predicted, one is weakly predicted and one is not predicted. These observations, together with the displayed intraspecies and interspecies variation clearly indicate the limitations of these predictive methods.

In conclusion, we have demonstrated that, despite the expected variation in the exact location of continuous epitopes defined by different anti‐rbGH sera, there are large regions of overlap defining immunogenic core regions within the molecule. We believe that studies of this nature, together with further understanding of antigen processing and peptide presentation to immune cells, may have a role to play in the development of candidate peptide vaccines.
Biological methane activation involves the intermediacy of carbon‐centered radicals
Authors: WILKINS Patricia C. , DALTON Howard , PODMORE Ian D. , DEIGHTON Nigel , SYMONS Martyn C. R.
Published: March 2005

The spin‐trapping technique has demonstrated that carbon‐centered radicals are produced during soluble‐methane‐monooxygenase catalysis of the hydroxylation of several different types of substrate. The resulting spin‐adducts were identified from the hyperfine splitting constants in their EPR spectra. Isotopic labelling showed unequivocally that the trapped radicals were derived from substrate. The carbon‐centered substrate radicals are believed to result from hydrogen‐atom abstraction by a ferryl species in a cytochrome‐P‐450‐like mechanism. No hydroxy radical nor an oxygen‐based radical of any kind was detected in any of the spin‐trapping experiments.
Aminopeptidase P from human leukocytes
Authors: RUSU Ileana , YARON Arieh
Published: March 2005

Cytosolic aminopeptidase P was obtained in highly purified form from human leukocytes by a four‐step procedure. Buffy coats were the starting material. A Mr of 140000 was obtained by size‐exclusion HPLC for the native enzyme. As shown by SDS/PAGE under reducing and denaturing conditions, the enzyme consisted of likely identical subunits with Mr of 71000. Purified aminopeptidase P cleaved off, specifically and efficiently, the N‐terminal residues from peptides with N‐terminal Xaa‐Pro sequences. The penultimate proline was not replaceable by hydroxyproline, alanine and glycine in di‐, tri‐ and tetrapeptides. Polyproline was not hydrolyzed. Dipeptides were cleaved (Arg‐Pro, Phe‐Pro > Trp‐Pro > Pro‐Pro) although slower than longer peptides. Cleavage was observed of several biologically active peptides; C‐terminal fragment (residues 201–206) of C‐reactive protein, oxytocin fragment Tyr‐Pro‐Leu‐Gly, morphiceptin, peptide Gly‐Pro‐Arg‐Pro (inhibitor of fibrin polymerization) and kentsin. In addition, cleavage of a protein, interleukin‐6, was also demon‐strated. Aminopeptidase P was maximally activated by Mn2+, and to a lesser extent by Co2+. The activity was optimal at pH 8. Ni2+, Zn2+ and especially Cd2+ caused marked inhibition. EDTA, 1,10‐phenantroline and dithiothreitol were also inhibitory. Carbobenzoxy‐phenylalanine, as well as several N‐carbobenzoxy‐proline‐containing peptides, caused partial inhibition. The observed resistance of Gly‐Pro, Pro‐Gly, Pro‐Ile to hydrolysis by the purified enzyme strongly indicates absence of known proline‐specific dipeptidases in the aminopeptidase‐P preparation.
Nuclear‐magnetic‐resonance analysis of the capsular antigen of Actinobacillus pleuropneumoniae serotype 9
Authors: BEYNON Linda M. , RICHARDS James C. , PERRY Malcolm B.
Published: March 2005

The specific capsular antigen of Actinobacillus pleuropneumoniae serotype 9 was characterized by one‐dimensional and two‐dimensional high‐field nuclear‐magnetic‐resonance methods, and by chemical analyses, as a teichoic‐acid‐type polymer of a repeating unit having the structure

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The basic polymer structure is identical to capsular antigens of Neisseria meningitidis group H, Escherichia coli K62 (K2ab) and Pasteurella haemolytica serotype T15.
Biosynthesis and metabolism of dipeptidylpeptidase IV in primary cultured rat hepatocytes and Morris hepatoma 7777 cells
Authors: LOCH Nikolaus , TAUBER Rudolf , BECKER Andreas , HARTEL‐SCHENK Sabine , REUTTER Werner
Published: March 2005

N‐Glycosylation, biosynthesis and degradation of dipeptidylpeptidase IV (EC 3.4.14.5) (DPP IV) were comparatively studied in primary cultured rat hepatocytes and Morris hepatoma 7777 cells (MH 7777 cells). DPP IV had a molecular mass of 105 kDa in rat hepatocytes and of 103 kDa in MH 7777 cells as assessed by SDS/PAGE under reducing conditions. This difference in molecular mass was caused by differences in covalently attached N‐glycans. DPP IV from hepatoma cells contained a higher proportion of N‐glycans of the oligomannosidic or hybrid type and therefore migrated at a slightly lower molecular mass. In both cell types DPP IV was initially synthesized as a 97‐kDa precursor which was completely susceptible to digestion with endo‐β‐N‐acetylglucosaminidase H converting the molecular mass to 84 kDa. The precursor was processed to the mature forms of DPP IV, glycosylated with N‐glycans mainly of the complex type with a half‐life of 20–25 min. The transit of newly synthesized DPP IV to the cell surface displayed identical or very similar kinetics in both cell types with the major portion of DPP IV appearing at the cell surface after 60 min. DPP IV molecules were very slowly degraded in hepatocytes as well as in hepatoma cells with half‐lives of approximately 45 h. Inhibition of oligosaccharide processing with 1‐deoxymannojirimycin led to the formation of DPP IV molecules containing N‐glycans of the oligomannosidic type. This glycosylation variant was degraded with the same half‐life as complex‐type glycosylated DPP IV. By contrast, inhibition of N‐glycosylation with tunicamycin resulted into rapid degradation of non‐N‐glycosylated DPP IV molecules in both cell types. Non‐N‐glycosylated DPP IV could not be detected at the cell surface indicating an intracellular proteolytic process soon after biosynthesis.
Isolation and functional analysis of histidine‐tagged elongation factor Tu
Authors: BOON Kathy , VIJGENBOOM Erik , MADSEN Lene V. , TALENS Anneke , KRAAL Barend , BOSCH Leendert
Published: March 2005

The study of the structure/function relationships of the Escherichia coli elongation factor Tu (EF‐Tu) via mutagenesis has been hampered by difficulties encountered in separating the mutated factor from other proteins, in particular native EF‐Tu. Here we describe a novel system for the purification of EF‐Tu mutant species, based on metal‐ion affinity chromatography. To facilitate rapid and efficient purification we designed a recombinant EF‐Tu with an additional C‐terminal sequence of one serine and six histidine residues. A cell extract containing the His‐tagged EF‐Tu (EF‐TuHis) is applied to a Ni2+ ‐nitrilotriacetic acid column. EF‐TuHis can be selectively eluted with an imidazole containing buffer, yielding a preparation of more than 95% purity, free of wild‐type EF‐Tu. In‐vitro and in‐vivo functional analyses show that EF‐TuHis resembles the wild‐type EF‐Tu, which makes this one‐step isolation procedure a promising tool for the study of the interactions of mutant EF‐Tu with the various components of the elongation cycle. The new isolation procedure was succesfully applied for the purification of a mutant EF‐TuHis with a Glu substitution for Lys237, a residue possibly involved in the binding of aminoacyl‐tRNA.
Synthetic peptides as antagonists of the anaphylatoxin C3a
Authors: KRETZSCHMAR Titus , POHL Martina , CASARETTO Monika , PRZEWOSNY Michael , BAUTSCH Wilfried , KLOS Andreas , SAUNDERS Derek , KÖHL Jörg
Published: March 2005

Peptide compounds resembling the receptor‐binding C‐terminal domain of the anaphylatoxic peptide C3a were synthesized to examine two kinds of C3a antagonism: (a) specific desensitization of C3a‐sensitive cells and (b) competitive binding to the C3a receptor. We used guinea‐pig platelets, which express a C3a receptor and specifically release ATP upon stimulation, to evaluate the actions of the C3a analogues. The ATP liberation can be inhibited by pretreatment (i.e. desensitization) of the guinea‐pig platelets with substimulatory concentrations of C3a or its analogues. Compared to C3a, several peptides were found with at least a tenfold greater difference between the required concentrations for C3a‐specific half‐maximal desensitization (DD50) and half‐maximal platelet activation (ED50). The most potent compounds were YAAALKLAR and Fmoc‐EAALKLAR (Fmoc: 9‐fluorenylmethoxycarbonyl) with an ED50/DD50 of 140 ± 28 and 80 ± 17, respectively (mean ± standard deviation). The ED50/DD50 of human C3a was found to be only 6 ± 2. Some C3a derivatives were also tested in competitive binding studies for their ability to compete with C3a for receptor sites on guinea‐pig platelets. Three of them were considered partial antagonists [YRRGRLAR, YRRGRXLAR and YRRGRXLAR (X=6‐aminohexanoyl)] because their Ki were smaller than their ED50 (Ki/ED50= 0.6 ± 0.3, 0.5 ± 0.1 and 0.4 ± 0.2, respectively). Interestingly, the last two compounds also had ED50/DD50 values greater than 60. Common to all three peptides are N‐terminal arginine‐rich sequences and intramolecular disulfide bridges which introduce conformational constraint.
β‐agonist‐induced inhibitory‐guanine‐nucleotide‐binding regulatory protein coupling to adenylate cyclase in mollusc Anodonta cygnea foot muscle sarcolemma
Authors: PERTSEVA Marianna N. , KUZNETZOVA Ludmila A. , PLESNEVA Svetlana A. , GRISHIN Alexander V. , PANCHENKO Mikhail P.
Published: March 2005

In the sarcolemma fraction of foot muscles of a fresh‐water bivalve mollusc, Anodonta cygnea, a direct inhibitory, rather than stimulatory, effect of the β‐adrenergic agonist isoproterenol, at micromolar concentration, on cAMP level and adenylate cyclase activity, was revealed. It was blocked by β‐ but not α‐adrenergic antagonists. A single class of [3H]dihydroalprenolol‐binding sites with binding properties of β‐adrenergic receptor was detected in mollusc sarcolemma. Potentiation of the inhibitory effect of isoproterenol on mollusc adenylate cyclase activity by GTP or guanosine 5′‐[β,γ‐imido]triphosphate at micromolar concentrations, and its elimination in the presence of guanosine 5′‐[β‐thio]diphosphate, were shown. The pertussis‐toxin‐induced ADP‐ribosylation of sarcolemma 40‐kDa protein [immunochemically related in the C‐terminal part to pertussis‐toxin‐sensitive guanine‐nucleotide‐binding regulatory protein (G‐protein) α subunits of vertebrates], as well as the treatment of mollusc sarcolemma with antisera responsive to the C‐terminus of vertebrate inhibitory G‐protein (Gi) α subunit led to elimination of the inhibitory effect of isoproterenol on adenylate cyclase activity. The results obtained suggest that β‐agonist‐induced inhibition of adenylate cyclase in A. cygnea foot muscle may be realized via the β‐adrenoreceptor/Gi signalling pathway.
Contribution of de‐novo and salvage synthesis to the uracil nucleotide pool in mouse tissues and tumors in vivo
Authors: ZAHAREVITZ Daniel W. , ANDERSON Lawrence W. , MALINOWSKI Nancy M. , HYMAN Roosevelt , STRONG John M. , CYSYK Richard L.
Published: March 2005

The relative contribution of de‐novo and salvage synthesis to tissue pyrimidine nucleotide pools is an important parameter in the rational design of anti‐pyrimidine therapies, but has not been measured in vivo. We have measured the contribution of de‐novo synthesis to the total acid‐soluble uracil nucleotide pool in mouse tissues by analysis of the incorporation of label after intra‐peritoneal infusion of L‐[15N]alanine. The contribution of salvage synthesis was measured by the incorporation of radiolabel after intravenous infusion of [14C]uridine. The results show that de‐novo synthesis makes the larger contribution to the intestine uracil nucleotide pool, salvage synthesis makes the larger contribution to the kidney pool, and de‐novo and salvage synthesis make roughly equal contributions to the liver pool. In tumors studied (L1210, P388, B16, Nettesheim), the contribution of de‐novo synthesis was at least five times the contribution of salvage synthesis. The measurements were repeated 24 hours after a 400‐mg/kg dose of N‐phosphonacetyl‐l‐aspartic acid. De‐novo synthesis was substantially inhibited in all tissues and tumors after this treatment, although significant residual activity was observed in the intestine and L1210 cells. Nettesheim carcinoma was the only tumor or tissue to show a significant increase in salvage synthesis after N‐phosphonacetyl‐l‐aspartic acid treatment.
Effect of 2‐mercaptoethanol on glutathione levels, cystine uptake and insulin secretion in insulin‐secreting cells
Authors: JANJIC Danilo , WOLLHEIM Claes B.
Published: March 2005

The role of glutathione (GSH) in the differentiated state of insulin‐secreting cells was studied using 2‐mercaptoethanol as a means of varying intracellular GSH levels. 2‐Mercaptoethanol (50 μM) caused a marked increase of GSH in two rat insulinoma cell lines, RINm5F and INS‐1, the latter being dependent on the presence of 2‐mercaptoethanol for survival in tissue culture. The effect of 2‐mercaptoethanol on GSH was shared by other thiol compounds. Since in other cell types 2‐mercaptoethanol is thought to act on cystine transport, thereby increasing the supply of cysteine for GSH synthesis, we have studied [35S]cystine‐uptake in INS‐1 cells. At equimolar concentrations to cystine, 2‐mercaptoethanol caused stimulation of [35S]cystine‐uptake. The effect persisted in the absence of extracellular Na+, probably suggesting the involvement of the Xc− carrier system. INS‐1 cells with a high GSH level, cultured 48 h with 2‐mercaptoethanol, displayed a lower cystine uptake than control cells with a low GSH content.

The effect of variations of the GSH levels on short‐term insulin release was studied. No alteration of glyceraldehyde‐induced or KCl‐induced insulin release in RINm5F cells was detected. In contrast, both in islets and in INS‐1 cells, a high GSH level was associated with a slighly lower insulin release. In INS‐1 cells the effect was more marked at low glucose concentrations, resulting in an improved stimulation of insulin secretion. On the other hand, in islets, a decrease in the incremental insulin release evoked by glucose was seen. As in other cell types, oxidized glutathione (GSSG) was less than 5% of total GSH, and in INS‐1 cells no change in the GSH/GSSG ratio was detected during glucose‐induced or 3‐isobutyl‐1‐methylxanthine‐induced insulin release.

In conclusion, 2‐mercaptoethanol‐dependent INS‐1 cells, as well as RINm5F cells and islets of Langerhans, display a low capacity in maintaining intracellular levels of GSH in tissue culture without extracellular thiol supplementation; 2‐mercaptoethanol possibly acts by promoting cyst(e)ine transport; changes in GSH levels caused a moderate effect on the differentiated function of insulin‐secreting cells.
The Soret magnetic circular dichroism of ferric high‐spin myoglobins
Authors: MATSUOKA Ariki , KOBAYASHI Nagao , SHIKAMA Keiji
Published: March 2005

To find a simple criterion for the presence of the distal (E7) histidine residue in myoglobins and hemoglobins, the Soret magnetic‐circular‐dichroic spectra were examined for ferric metmyoglobins from various species. A distinct and symmetric dispersion‐type curve was obtained for myoglobins containing the distal histidine, whereas a relatively weak and unsymmetric pattern was observed for myoglobins lacking this residue, such as those from three kinds of gastropodic sea molluscs, a shark and the African elephant. The magnetic‐circular‐dichroic spectra obtained would thus be a direct reflection of the presence or absence of a water molecule at the sixth coordinate position of the heme iron(III), this axial water ligand being stabilized by hydrogen‐bond formation to the distal histidine residue. On the basis of these Soret magnetic‐circular‐dichroic signals, we also examined the structure of a protozoan myoglobin (or a monomeric hemoglobin) from Paramecium caudatum of particular interest for the evolution of these proteins from protozoa to higher animals.
Mechanism of carboxypeptidase‐Y‐catalysed peptide semisynthesis
Authors: CHRISTENSEN Ulla , DRØHSE Helle B. , MØLGAARD Lone
Published: March 2005

The initial rate steady‐state kinetics of carboxypeptidase‐Y‐catalyzed hydrolysis and aminolysis reactions with some α‐N‐benzoyl‐l‐tyrosinyl compounds has been investigated using L‐valinamide as the nucleophile in aminolysis. Hydrolysis of α‐N‐benzoyl‐l‐tyrosinyl ethyl ester, 4‐nitroanilide, and‐amide has been studied in the pH range 4–9. The results are interpreted in terms of the classical serine proteinase mechanism, which involves enzyme‐substrate complex formation, followed by acylation and deacylation of the enzyme. The three reactions share the same deacylation step. It is rate‐determining with the ester substrate, but with the 4‐nitroaniline acylation is and this is even more pronounced with the amide. From the pH dependencies, no change of rate‐determining step is apparent in the range pH 4–9. For the 4‐nitroanilide and the amide substrates, the kinetic paramcter, kc/Km, is influenced by an ionizing group with a pK value of 6. Probably this is the active‐site histidine residue, which thus is active in acylation in its deprotonated form. That group affects the deacylation reaction similarly as seen from the kinetics of the ester substrate.

Aminolysis occurs in parallel to hydrolysis in the presence of reactive nucleophiles. Here L‐valinamide was used as model nucleophile. The analysis of the observed kinetic effects of L‐valinamide on the initial rate behaviour of carboxypeptidase‐Y‐catalyzed hydrolysis reactions suggests a reaction mechanism which involves (a) the binding of the free nucleophile to the free enzyme and (b) reaction of the free nucleophile with the acyl‐enzyme complex forming an enzyme‐aminolysis product complex, which dissociates into the free enzyme and the aminolysis product. The reactions are characterized by a number of kinetic parameters, the values of which are determined.

The results of aminolysis progress reactions indicate that the formation of the product in high yields is strongly dependent on the leaving group of the substrates. The initial production of aminolysis product, however, is the same for the three substrates. But the fact that their kc/Km values differ by several orders of magnitude leads to significantly different progresses of the aminolysis. The ester substrate is the only one that efficiently competes with and hinders the hydrolysis of the aminolysis product.
Purification and properties of a novel arylmalonate decarboxylase from Alcaligenes bronchisepticus KU 1201
Authors: MIYAMOTO Kenji , OHTA Hiromichi
Published: March 2005

A novel decarboxylase which catalyzes an enantioselective decarboxylation of α‐aryl‐α‐methylmalonates to α‐arylpropionates has been purified from a soil bacterium Alcaligenes bronchisepticus KU 1201. The enzyme was purified 300‐fold to homogeneity, judged from the analysis of N‐terminal amino acid sequence, and found to be a monomeric enzyme of apparent 24 kDa. The enzyme catalyzes a decarboxylation giving α‐arylalkanoates from substituted malonates such as α‐arylmalonate and α‐alkyl‐α‐arylmalonates. The decarboxylase is not a biotin containing enzyme because avidin have no influence on the enzyme activity. In addition, the enzyme does not require known co‐factors (ATP, ADP and coenzyme A) for maximum activity. The enzyme activity was inhibited by sulfhydryl agents. The electronic effect of the substituents on kcat for the enzymic decarboxylation of arylmalonates has been studied. The logarithm of relative value of kcat gave a linear correlation to Hammett's σ with a δ value of +1.9, for substituted phenylmalonates. Comparing the relative activities, it is clear that the enzyme prefers α‐arylmalonates to α‐aryl‐α‐methylmalonates. Thus, the enzyme was tentatively named as arylmalonate decarboxylase.
Identification, purification and characterization of a novel phosphatidylinositol‐specific phospholipase C, a third member of the β subfamily
Authors: CAROZZI Amanda J. , KRIZ Ron W. , WEBSTER Catherine , PARKER Peter J.
Published: March 2005

The partial sequence of a novel PtdIns‐specific phospholipase C of the β subfamily (PtdIns‐PLCβ3) is described. Based upon the predicted protein sequence, monospecific antibodies have been raised and used to identify a suitable source for purification of the protein. Fractionation of HeLa S3 cells revealed that immunoreactive PtdIns‐PLCβ3 is membrane associated; purification (∼1000‐fold) from this fraction yielded a single immunoreactive protein of 158 kDa, with a specific activity of 136 μmol · min−1· mg−1, with PtdIns 4,5‐bisphosphate as substrate. Substrate specificity and Ca2+ dependence of this purified PtdIns‐PLC are characteristic of the PtdIns‐PLCβ subfamily.
Molecular mimicry of trifluoroacetylated human liver protein adducts by consitutive proteins and immunochemical evidence for its impairment in halothane hepatitis
Authors: GUT Josef , CHRISTEN Urs , HUWYLER Jörg , BÜRGIN Maria , KENNA J. Gerald
Published: March 2005

A monospecific antibody (anti‐CF3CO antibody) was obtained by affinity chromatography on a Nɛ‐trifluoroacetyl‐l‐lysine (CF3CO‐Lys) matrix of a rabbit polyclonal antiserum, directed against trifluoroacetylated protein adducts (CF3CO‐proteins). The anti‐CF3CO antibody recognized distinct CF3CO‐proteins on immunoblots of a liver biopsy obtained from a human individual 10 h after halothane anaesthesia. Cross‐reactive proteins of 52 kDa and 64 kDa were recognized on immunoblots of livers obtained from human individuals not exposed to halothane. Recognition of both CF3CO‐proteins and the 52‐kDa and 64‐kDa cross‐reactive proteins was abolished in the presence of 1 mM CF3CO‐Lys. Anti‐CF3CO antibody, affinity‐adsorbed to the 52‐kDa or the 64‐kDa cross‐reactive proteins of human liver, recognized the majority of target CF3CO‐proteins on immumoblots of the human liver biopsy of an individual exposed to halothane. Liver biopsies of 5 out of 7 (71%) patients with halothane hepatitis exhibited an absence or low amounts of immuno‐recognizable 52‐kDa and/or 64‐kDa cross‐reactive proteins. In contrast, of 22 control human individuals tested, all liver tissue samples were positive for the 52‐kDa and/or the 64‐kDa cross‐reactive proteins. These data indicate that epitopes on the cross‐reactive proteins of 52 kDa and 64 kDa of human liver bear strong immunochemical resemblance to epitopes on human liver CF3CO‐proteins. Low‐level expression of the cross‐reactive proteins of 52 kDa and 64 kDa is discussed as one possible factor in human susceptibility to halothane hepatitis.
Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush‐border membranes from mosquito larvae
Authors: NIELSEN‐LEROUX Christina , CHARLES Jean‐François
Published: March 2005

The presence of specific receptors for Bacillus sphaericus binary toxin on brush‐border membrane fractions (BBMF) from Culex pipiens larvae midgut cells was demonstrated by an in vitro binding assay. Both activated and radiolabelled polypeptides from the 51‐kDa and 42‐kDa binary toxin of B. sphaericus 1593 specifically bound to BBMF. Direct binding and homologous competition experiments indicated a single class of B. sphaericus toxin receptors, with a dissociation constant (Kd) of approximately 20 nM and a maximum binding capacity (Bmax) of approximately 7 pmol/mg BBMF protein. The sugars GalNAc, GlcNAc and N‐acetyl neuraminic acid had no detectable inhibitory effect on toxin binding to C. pipiens BBMF. Binding experiments with the non‐susceptible mosquito species Aedes aegypti failed to detect significant binding of B. sphaericus binary toxin to A. aegypti BBMF.
The kinetic mechanism of sheep liver sorbitol dehydrogenase
Authors: LINDSTAD Rune I. , HERMANSEN Leonila F. , McKINLEY‐McKEE John S.
Published: March 2005

The relations between the kinetic parameters for both sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase show that a Theorell‐Chance compulsory order mechanism operates from pH 7.4 to 9.9 This is supported by many parallels with the kinetics of horse liver alcohol dehydrogenase, which operates by this classical mechanism.

An isotope‐exchange study using D‐(2H8)sorbitol confirmed the existence of ternary complexes and that, under maximum velocity conditions, their interconversion is not rate‐determining. Substrate inhibition at high concentrations of D‐sorbitol or D‐fructos confirmed rate‐determining enzyme—coenzyme product dissociation, slowed by the existence of more stable abortive ternary enzyme‐conenzyme product complexes with substrate. The effect of the inhibitor/activator 2,2,2‐tribromo‐ethanol showed the existence of enzyme‐NAD‐CBr3CH2OH complexes inhibiting the first phase of reaction and enzyme‐NADH‐CBr3CH2OH complexes dissociating more rapidly than the usual rate‐determining enzyme‐NADH coenzyme product dissociation in the final phase. Inhibition studies with dithiothreitol also confirmed an ordered binding of coenzymes and second substrates to sorbitol dehydrogenase. Neither D‐sorbitol nor D‐fructose had any effect on enzyme inactivation by the affinity labelling reagent DL‐2‐bromo‐3‐(5‐imidazolyl)propionic acid, thus giving no evidence for their existence as binary enzyme‐substrate complexes.

Several alternative polyol substrates for sorbitol dehydrogenase gave the same maximum velocity as sorbitol. This indicated a common rate‐limiting binary enzyme‐NADH product dissociation and a similarity of mechanism.

An enzyme assay for pH 7.0 and 9.9 is given which enables the concentration of sorbitol dehydrogenase to be determined from initial rate measurements of enzyme activity.
Assignment of the magnetic resonances of the imino protons and methyl protons of Bombyx mori tRNAGCCGly and the effect of ion binding on its structure
Authors: AMANO Mayumi , KAWAKAMI Makoto
Published: March 2005

The magnetic resonances in the low‐field H‐NMR spectra of Bombyx mori tRNAGCCGly, corresponding to the hydrogen‐bonded imino protons of the helical stems and tertiary base pairs, could be tentatively assigned by means of the sequential nuclear Overhauser effects. While B. mori tRNAGCCGly does not contain the G19C56 tertiary base pair, the D20G57 base pair exists between the D and T loops, which was not found in the X‐ray crystal structure of yeast tRNAPhe. The effects of Mg2+, spermine and temperature on the conformation of this tRNA have also been examined based on the behavior of the assigned resonance signals. Mg2+ stabilize the D and T stems and the tertiary structure between the D and T loops. Spermine affects the resonances of the D and anticodon stems, and A23G9, but does not stabilize them. While the acceptor stem melts sequentially from both ends (G7C66 and G1C72) with increasing temperature, the anticodon stem melts from only one end (G39C31) and the G26C44 base pair is the most stable. In the tertiary structure between the variable loop and D stem, G10G45 melts first and G22G46 last. Yeast tRNAPhe has also been examined, and the results were compared with those for B. mori tRNAGly.
1H‐NMR assignments and local environments of aromatic residues in bovine, human and guinea pig variants of α‐lactalbumin
Authors: ALEXANDRESCU Andrei T. , BROADHURST R. William , WORMALD Claire , CHYAN Chia‐Lin , BAUM Jean , DOBSON Christopher M.
Published: March 2005

1H‐NMR assignments have been defined for the aromatic‐ring protons of the bovine, guinea pig and human variants of α‐lactalbumin. Spin‐system networks were identified by means of double‐quantum‐filtered two‐dimensional J‐correlated spectroscopy and two‐dimensional relayed coherence spectroscopy data. Analysis of two‐dimensional nuclear‐Overhauser‐enhancement spectroscopy data of the proteins indicated that in each case two clusters of aromatic residues exist. The two clusters are also evident in the crstal structure of the human protein, and this evidence, in conjunction with sequence differences between the three proteins, permitted sequence‐specific assignments to be made for the majority of aromatic residues. Remaining ambiguities in the assignments could be resolved by analysis of photochemically induced dynamic nuclear polarization (PCIDNP) effects. Comparison of the PCIDNP spectra of the three proteins indicated the presence of only minor differences in the surface exposure of conserved aromatic residues. Taken together, these results indicate that the environments of the conserved aromatic residues in bovine, guinea pig and human α‐lactalbumin in solution are very similar to each other, and that the solution and the crystal forms of at least the human protein are similar.
Catalysis by α‐chymotrypsin entrapped into surface‐modified polymeric nanogranules in organic solvent
Authors: KHMELNITSKY Yuri L. , NEVEROVA Irina N. , GEDROVICH Alexey V. , POLYAKOV Vladimir A. , LEVASHOV Andrey V. , MARTINEK Karel
Published: March 2005

Physicochemical characteristics of previously suggested surface‐modified polymeric nanogranules (SMPN) and catalytic and stability properties of α‐chymotrypsin entrapped into such nanogranules in a nonpolar solvent were investigated in more details. SMPN were obtained by polymerization of an acrylamide/N, N'‐methylene‐bisacrylamide mixture in a mixed reversed micellar system composed of Aerosol OT [sodium di(2‐ethylhexyl)sulfosuccinate] and the polymeric surfactant Pluronic F‐108 modified with polymerizable groups, followed by the chromatographic removal of the auxiliary surfactant, Aerosol OT. An optimal solvent system was found providing the required orientation of the polymeric surfactant in starting mixed micelles, i.e. with polar fragments immersed into the micellar interior and apolar fragments protruding into organic solvent. The hydrodynamic diameter of SMPN in benzene solution was estimated by means of quasi‐elastic light scattering to be 84 ± 1 nm. Catalytic and stability properties of α‐chymotrypsin entrapped into SMPN strongly depended on conditions of preparation of SMPN. The optimal concentration of acrylamide monomers in the micellar interior and hydration degree of starting reversed micelles were found to be 20% by mass and Wo= 15, respectively. α‐Chymotrypsin‐containing SMPN were used as a catalyst in the synthesis of N‐acetyl‐l‐tyrosine ethyl ester from N‐acetyl‐l‐tyrosine and ethanol, performed in a membrane reactor.
Decreased stress inducibility of the HSP68 protein in a rat hepatoma variant clone
Authors: PIRITY Melinda , NGUYEN Van Trung , DUBOIS Marie Francoise , BENSAUDE Olivier , HEVÉR‐SZABÓ Anna , VENETIANER Anikó
Published: March 2005

Analysis of the stress response of closely related rat hepatoma clones revealed that the major inducible heat‐shock protein 68 (HSP68) was only slightly inducible upon stress in the glucocorticoid‐resistant, dedifferentiated clone‐2 cells, but strongly activated in the differentiated, glucocorticoid‐sensitive Faza 967 cells from which clone 2 was derived. The decreased inducibility of HSP68 in clone‐2 cells was not the consequence of altered kinetics of protein synthesis recovery, was not correlated with the deficient inducibility of other major heat‐shock proteins and had no effect on the heat sensitivity of the cells. This deficiency was observed after treatment with mild and strong heat and various chemicals. The results of nuclear run‐on experiments suggested that the impairment of HSP68 mRNA induction most likely occurs at the transcriptional level and is probably specific for the corresponding gene. In Faza 967 and clone‐2 cells, stress activated comparable levels of heat‐shock‐factor binding to the heat‐shock element, and the expression of a reporter gene under the control of murine HSP70.1 promoter was strongly stimulated in both cells. Therefore, our results raise the possibility that the deficient stress inducibility of HSP68 is due to some specific regulation of the endogeneous HSP68 gene, rather than to a deficiency of the heat‐shock factor or mutation of the corresponding gene.
A chick skeletal‐muscle α‐actinin gene gives rise to two alternatively spliced isoforms which differ in the EF‐hand Ca2+‐binding domain
Authors: PARR Timothy , WAITES Gillian T. , PATEL Bipin , MILLAKE David B. , CRITCHLEY David R.
Published: March 2005

A chick non‐muscle α‐actinin cDNA probe encoding the EF‐hand region of molecule was used to screen a λgt10 chick brain cDNA library from 14‐day embryos. A partial 2.1‐kb α‐actinin cDNA was isolated (8W cDNA) which encoded a protein identical to chick skeletal‐muscle α‐actinin, except in the C‐terminal part of the first EF hand. In the variant, the 22 residues found in the skeletal‐muscle isoform were replaced by a stretch of 26 unique residues. Analysis of the structure of the skeletal‐muscle α‐actinin gene showed that the region of divergence was encoded by two exons which are alternatively spliced. Quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) was used to investigate the levels of the α‐actinin transcripts in various tissues. The skeletal‐muscle α‐actinin variant was expressed at low levels in brain, liver and spleen, but could not be detected in skeletal muscle. Surprisingly, skeletal‐muscle α‐actinin mRNA was also expressed in brain, liver and spleen. The RT/PCR products were authenticated by using diagnostic restriction enzyme sites and by sequencing. The splice variant derived from the skeletal‐muscle α‐actinin gene was also detected in a variety of cDNA libraries from both adult and embryonic tissues by PCR. Although a transcript encoding this α‐actinin splice variant is expressed in non‐muscle tissues, neither of the two EF‐hands would be predicted to be functional, making it unlikely to be a typical non‐muscle isoform which are calcium‐sensitive with respect to binding actin. The two vertebrate non‐muscle α‐actinins sequenced to date also have a spacer of five amino acids between the two EF hands, whereas in the variant, the spacer is just four residues in length. Further analysis will be required before this α‐actinin isoform, which we refer to as SKv, can be classified as muscle or non‐muscle α‐actinin. We propose a new nomenclature to describe the various α‐actinin genes and their transcripts.
Amino‐acid sequence and cell‐adhesion activity of a fibril‐forming collagen from the tube worm Riftia pachyptila living at deep sea hydrothermal vents
Authors: MANN Karlheinz , GAILL Francoise , TIMPL Rupert
Published: March 2005

We have determined the amino acid sequence of the α chain of a fibril‐forming collagen from the body wall of the marine invertebrate Riftia pachyptila (vestimentifera) by Edman degradation. The pepsin‐solubilized collagen chain consists of a 1011‐residue triple‐helical domain and short remnants of N‐ and C‐telopeptides. The triple‐helical sequence showed one imperfection of the collagen Gly‐Xaa‐Yaa triplet repeat structure due to a Gly Ala substitution. This imperfection imperfection is correlated to a prominent kink in the molecule observed by electron microscopy. No strong sequence similarity was found with the fibril‐forming vertebrate collagen types I‐III, V and XI except for the invariant Gly residues. However, one of the two consensus cross‐linking sequences was well conserved. The Riftia collagen shared with the vertebrate collagens many post‐translational modifications. About 50% of the Pro and Lys residues are found in the Yaa position and were extensively hydroxylated to 4‐hydroxyproline (4Hyp) and hydroxylysine (Hyl). A few proline residues in Xaa position were partially hydroxylated to either 4Hyp or 3Hyp. Despite the low sequence similarity, Riftia collagen was a potent adhesion substrate for two human cell lines. Cell adhesion could be inhibited by antibodies against the integrin β1 subunit but not by RGD peptides. This biological activity is apparently conserved in fibril‐forming collagens of distantly related species but does not require the two RGD sequences present in Riftia collagen.
Purification and characterization of glycerate kinase from a serine‐producing methylotroph, Hyphomicrobium methylovorum GM2
Authors: YOSHIDA Toyokazu , FUKUTA Kazuhiro , MITSUNAGA Toshio , YAMADA Hideaki , IZUMI Yoshikazu
Published: March 2005

The glycerate kinase of a serine‐producing methylotroph, Hyphomicrobium methylovorum GM2. was purified to complete homogeneity and characterized, the first time for an enzyme from a methylotroph. The enzyme was a monomer with a molecular mass about 41–52 kDa. The enzyme was stable against heating at 35°C for 30 min at pH values over 6–10. Maximum activity was observed at pH 8.0 and around 50°C. The Km values for D‐glycerate and ATP were 0.13 mM and 0.13 mM, respectively. The enzyme showed high specificity for D‐glycerate, and was activated by potassium and ammonium ions. The reaction product of the enzyme was identified as 2‐phosphoglycerate.
Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state
Authors: BLOTNICK Edna , MUHLRAD Andras
Published: March 2005

The structure of myosin subfragment 1 (S1) in the weakly attached complex with actin was studied at three specific sites, at the 50‐kDa/20‐kDa and 27‐kDa/50‐kDa junctions, and at the N‐terminal region, using tryptic digestion as a structure‐exploring tool. The structure of S1 at the vicinity of the 50‐kDa/20‐kDa junction is pH dependent in the weakly attached state because the tryptic cleavage at this site was fully protected by actin at pH 6.2, but the protection was only partial at pH 8.0. Since the actin protection is complete in rigor at both pH values, the results indicate that the structure of S1 at the 50‐kDa/20‐kDa junction differs in the two states at pH 8.0, but not at pH 6.2. Actin restores the ADP‐suppressed tryptic cleavage after Lys213 at the 27‐kDa/50‐kDa junction in the strongly attached state, but not in the weakly attached state, which indicates structural difference between the two states at this site. ATP and ADP open a new site for tryptic cleavage in the N‐terminal region of the S1 heavy chain between Arg23 and Ile24. Actin was found to suppress this cleavage in both weakly and strongly attached states, which shows that, in the vicinity of this site, the structure of S1 is similar in both states. The results indicate that the binding of S1 to actin induces localized changes in the S1 structure, and the extent of these changes is different in the various actin‐S1 complexes.
The uncoupling protein from brown adipose tissue mitochondria
Authors: VIGUERA Ana Rosa , GOÑI Félix M. , RIAL Eduardo
Published: March 2005

The uncoupling protein from brown adipose tissue is a member of the family of metabolite carriers of the mitochondrial inner membrane. It contains two tryptophan residues which have been characterized by fluorescence spectroscopy. Application of fluorescence‐quenching‐resolved spectroscopy (FQRS) allowed the determination of the emission maximum for each residue, both of which occur at 332 nm, thus suggesting that they are both located in a non‐polar environment. Fluorescence quenching has demonstrated that both residues are accessible to acrylamide and inaccessible to Cs+, while only one of them is accessible to I−. When FQRS is combined with guanidinium hydrochloride denaturation, the unfolding of the regions containing each tryptophan can be monitored separately as they are transferred to the polar medium where the emission maximum appears at 359 nm, revealing also that the iodide‐accessible residue is more sensitive to the denaturant. Secondary structure predictions, together with the data presented here, suggest that the iodide‐accessible residue could correspond to Trp173 and the denaturant‐resistant iodide‐inaccessible one to Trp280, located in the center of the sixth transmembrane α‐helix. Interaction of the protein with GDP (a transport inhibitor) has been studied and has revealed that it partially shields Trp173 from the interaction with I−, as well as reducing the static component of the acrylamide quenching.
The primary structure of piscine (Oncorhynchus mykiss) retinol‐binding protein and a comparison with the three‐dimensional structure of mammalian retinol‐binding protein
Authors: ZAPPONI Maria Carla , ZANOTTI Giuseppe , STOPPINI Monica , BERNI Rodolfo
Published: March 2005

The primary structures of two variants of rainbow trout (Oncorhynchus mykiss) plasma retinol‐binding protein (RBP) were determined and found to be approximately 60% identical with those of both human and Xenopus laevis RBPs. The comparable sequence similarities that we have found agree with the estimate of similar divergence times between bony fishes and mammals and between bony fishes and amphibians. The two piscine RBP variants differ by six amino acid substitutions at positions that are not crucial for the interaction with retinol, on the basis of the human RBP three‐dimensional structure [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1990) Proteins Struct. Func. Genet. 8, 44–61].

2. Models were developed for the three‐dimensional structures of rainbow trout and X. laevis RBPs, based on that of human RBP. The overall three‐dimensional structure appears to be very well preserved for RBPs isolated from vertebrate species for which the divergence time is 350–400 million years. At variance with an almost absolute conservation for the residues that participate in the formation of the retinol binding site in mammalian RBPs, several amino acid replacements are found for this part of the RBP molecule when the comparison is extended to piscine and amphibian RBPs. However, the only allowed amino acid replacements are either conservative or more than 0.4 nm distant from retinol. Besides the retinol binding site, a few regions at the protein surface appear to be rather conserved during phylogenetic development of vertebrates and, therefore, might be involved in molecular interactions.
Time‐resolved fluorescence study of human recombinant interferon α2
Authors: VINCENT Michel , LI DE LA SIERRA Ines M. , BERBERAN‐SANTOS Mário N. , DIAZ Aída , DIAZ Maylin , PADRON Gabriel , GALLAY Jacques
Published: March 2005

Human recombinant interferon α2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited‐state‐lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation‐time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited‐state‐lifetime distribution which becomes more heterogeneous. Disulfjtiebond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence‐intensity decay with four excited‐state‐lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast‐anisotropy‐decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady‐state and time‐resolved fluorescence‐anisotropy measurements in vitrified medium [95% (by mass) glycerol at ‐ 38°C]. This suggestion is in agreement with the recently published three‐dimensional structure of the homologous protein murine interferon β [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193–3201].
The hemoglobins of Notothenia angustata, a temperate fish belonging to a family largely endemic to the Antarctic Ocean
Authors: FAGO Angela , D'AVINO Rossana , DI PRISCO Guido
Published: March 2005

The blood of the teleost Notothenia angustata contains a major hemoglobin (Hb 1, over 95% of the total), accompanied by a minor component (Hb 2). The two hemoglobins have identical β chains and differ in their α chains. The primary structure of both hemoglobins has been established through the elucidation of the complete amino acid sequence of the three chains. The study of the oxygen‐binding properties shows that Hb 1 displays the Bohr and Root effects and has high affinity for organic phosphates.

N. angustata belongs to the family Nototheniidae, suborder Notothenioidei. Unlike the vast majority of nototheniid species, which live in isolation in the Antarctic Ocean and have developed cold adaptation, N. angustata inhabits the waters of southern New Zealand and is not cold adapted. Although some hematological parameters typically favour oxygen transport in a temperate environment, the hemoglobin multiplicity and structural and functional features closely resemble those of the Antarctic species of the same family and suborder. Thus, N. angustata may be considered as a link between temperate and Antarctic habitats. The hypothetical separation history of N. angustata from the Antarctic species of the same family is discussed in the light of the present findings.