bluuebunny/crossref_metadata_2025_split huggingface dataset:
Removal of N‐acetyl groups from blocked peptides with acylpeptide hydrolase
Authors: FARRIES Timothy C. , HARRIS Alan , AUFFRET Anthony D. , AITKEN Alastair 
Published: March 2005

Acylpeptide hydrolase, an enzyme that removes the modified residue from N‐terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross‐linking with dimethyl‐suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N, N‐dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis.Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non‐acetylated peptides are then adsorbed out with isothiocyanato‐glass. The N‐terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N‐terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide.
The proteinase yscB inhibitor (PBI2) gene of yeast and studies on the function of its protein product
Authors: SCHU Peter , SUAREZ RENDUELES Paz , WOLF Dieter H. 
Published: March 2005

The gene for proteinase yscB inhibitor I2B (PBI2) from Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library, and was sequenced. The gene codes for a single protein of 75 amino acids. In contrast to the published amino acid sequence [Maier, K., Müller, H., Tesch, R., Trolp, T., Witt, I. & Holzer, H. (1979) J. Biol. Chem. 254, 12555–12561] the DNA sequence revealed a valine instead of a leucine at position 33 (32 of the mature protein). Therefore the primary sequences of the isoinhibitors I2B of S. cerevisiae and I1B of Saccharomyces carlsbergensis differ only at position 34 (glutamic acid/lysine).The open reading frame of PBI2 was replaced in vitro by the URA3gene and a I2B null mutant of S. cerevisiae was constructed by gene replacement. The mutation resulted in an elevation of the protein degradation rate by 50% when grown under nutritional stress compared to the isogenic wild type. Growth and viability of the cells was not significantly affected by the absence of I2B.
Possible regulation of hypothalamus and lung histidine decarboxylase activity by cAMP‐dependent protein kinase
Authors: HUSZTI Zsuzsanna , MAGYAR Kálmán , KELETI Judith 
Published: March 2005

Activity of crude histidine decarboxylases (HisDC) from the hypothalamus and the lungs, was markedly reduced by incubating with ATP. Mg, cAMP and cAMP‐dependent protein kinase A, whereas activity of the crude glandular stomach enzyme changed only slightly under equal condition. The omission of one of these components failed to reduce HisDC activity by as much as the complete system. Addition of bovine heart (type II) or rat cerebellum protein kinase A (types I and II) inhibitor to the assay prevented enzyme inactivation; moreover, protein kinase A inhibitors permitted moderate activation under phosphorylating and control conditions.Cytosolic hypothalamus HisDC activity was elevated 2—2.2‐fold by incubating the cytosol for 15 min in the presence of MnCl2, a known stimulator of phosphoprotein phosphatase; this was prevented when 20 mM NaF, a common inhibitor of phosphoprotein phosphatase, was added to the cytosol.The apparent Km of ATP. Mg‐treated hypothalamus HisDC for histidine was elevated 5—10‐fold compared to controls, whereas the Vmax was approximately the same. Under this condition, the Km was calculated as high as 0.5—2.2 mM (depending on phosphorylating conditions), while controls had a Km of 0.1—0.3 mM (depending on the initial phosphorylating states). Addition of rabbit muscle (type I), bovine heart (type II) or rat cerebellum (types I and II) inhibitor of protein kinase A, to the phosphorylating mixture, abolished the difference in Km between control and ATP. Mg‐treated HisDC. Moreover, rat cerebellum protein kinase A inhibitors increased Vmax to above the control level; while 20 mM NaF (inhibitor of phosphoprotein phosphatase) decreased Vmax to approximately one half of that of the controls.These data indicate that HisDC activity in the hypothalamus and the lungs, but not in the stomach, is affected in oppositely by protein kinase A and phosphoprotein phosphatases.
Binding of long‐chain fatty acids to serum albumin in healthy humans Relationship to obesity
Authors: BRODERSEN Rolf , VORUM Henrik , KRUKOW Niels , PEDERSEN Anders Overgaard 
Published: March 2005

Equilibria of the binding of palmitate to serum albumin in adults are studied by dialysis‐exchange‐rate determinations. The results are used for a description of binding equilibria of fatty acids in general, as follows.1. The reserve albumin concentration, p, for binding of palmitate is used as an approximate measure of p*, the reserve albumin concentration for binding of mixed fatty acids present in serum.2. The total availability of fatty acids is defined as C*/p*, where C* is the total concentration of non‐esterified fatty acid.3. The fatty‐acid‐binding property of albumin is described by L*=p*/P—αC*/P is the albumin concentration. The numerical value of α is −0.05.The above parameters are measured in sera from four healthy volunteers, in whom large variations of serum fatty acid concentration occurred. A group of 64 healthy students showed considerable variation of L* from one individual to another. It is found that L* decreases significantly with increasing body mass index (body mass divided by the square of the body length). In 42 patients with diabetes type I, L* was independent of body mass index. These findings are consistent with a previously formulated hypothesis of the mechanism of obesity.
A study on the inactivation of wheat carboxypeptidase
Authors: KUNUGI Shigeru , TANABE Kazuo , KONDOH Toshihiro , YOKOYAMA Masahito , ITO Nobuya , NOMURA Akihiko , FUKUDA Mitsuhiro 
Published: March 2005

The mechanism of inactivation of carboxypeptidase from wheat at high pH was studied kinetically and spectrophotometrically.Inactivation of wheat carboxypeptidase is characterized by initial, transient high activity, soon followed by loss of activity, accompanied by an increase in both fluorescence intensity and anisotropy and a decrease a decrease in circular dichroism.A scheme was proposed in which the enzyme undergoes dissociation into monomers, the total activity of which becomes twice that of the normal dimer on a mass basis, and soon further denatures and aggregates.Treatment of the enzyme with a bifunctional reagent partly prevented denaturation of the monomer and hence increased the peptide synthetic activity.
Molecular modelling of bacterial deep rough mutant lipopolysaccharide of Escherichia coli
Authors: KASTOWSKY Manfred , SABISCH Andreas , GUTBERLET Thomas , BRADACZEK Hans 
Published: March 2005

Molecular modelling techniques have been applied to compute the conformations accessible to bacterial deep rough lipopolysaccharide of Escherichia coli (Re‐LPS). Analyses of the results showed that the models typically exhibit a tilt of the diglucosamine backbone with respect to the membrane normal of 53 ± 7° while both the glucosamine ring planes are orientated approximately parallel to the membrane normal. Different models were found to show compact and elongated types of acyl chain arrangements, both producing anisotropic lateral dimensions of the models of 1.0–1.1 nm and 1.7–2.0 nm for the shorter and the longer side, respectively. The conformationally allowed range of the isolated dOclA(α‐2‐4)dOclA disaccharide (dOclA = 3‐deoxy‐d‐manno‐octulosonic acid) was found to be extremely limited. It appeared that the dOclA disaccharide (dOclA)2 is centred at the top of the Re‐LPS molecule preferring two orientations stabilized by hydrogen bonds involving only one phosphate group of the lipid A moiety at a time.The effect of charges on the Re‐LPS conformations has been studied in separate calculations. From these calculations it was obvious that charges have no significant effects on the conformations of the isolated lipid A and (dOclA)2 moieties. However, it was found that the orientation of (dOclA)2 with respect to the lipid A part is highly sensitive to charges, i.e. in the charged models the proximity of phosphate and carboxyl groups is prevented by strong electrostatic repulsion between these negatively charged groups.In order to rationalize the acyl chain packing of the models, a simple geometrical model which correlates the tilt of the diglucosamine backbone with the energetically favoured close packing of the acyl chains is proposed. Furthermore, the possibility of a chelate‐like complexation of divalent cations and its contribution to head group mobility is discussed.
Nuclear transition protein 1 from ram elongating spermatids
Authors: CHIRAT Frédéric , MARTINAGE Arlette , BRIAND Gilbert , KOUACH Mostafa , VAN DORSSELAER Alain , LOIR Maurice , SAUTIÈRE Pierre 
Published: March 2005

The ram transition protein 1 (TP1) is present in spermatid cell nuclei in the nonphosphorylated, monophosphorylated and diphosphorylated forms. Its primary structure was determined by automated Edman degradation of S‐carboxamidomethylated protein and of peptides generated by cleavage with thermolysin and endoproteinase Lys‐C.The ram TP1 is a small basic protein of 54 residues and structurally very close to other mammalian TP1. The mass spectrometric data obtained from the protein and its fragments reveal that ram TP1 is indeed a mixture (approximately 5:1) of two structural variants (Mr 6346 and 6300). These variants differ only by the nature of the residue at position 27 (Cys in the major variant and Gly in the minor variant). The study of phosphorylation sites has shown that four different serine residues could be phosphorylated in the monophosphorylated TP1, at positions 8, 35, 36 or 39.From previous physical studies, it has been postulated that the Tyr32 surrounded by two highly conserved basic clusters was responsible for the destabilization of chromatin by intercalation of its phenol ring between the bases of double‐stranded DNA.The presence of three phosphorylatable serine residues in the very conserved sequence 29–42 is another argument for the involvement of this region in the interaction with DNA.
Purification and identification of 91‐kDa neutrophil gelatinase
Authors: MASURE Stefan , PROOST Paul , VAN DAMME Jo , OPDENAKKER Ghislain 
Published: March 2005

Human neutrophils were found to release a 91‐kDa gelatinase that is serologically related to tumor‐derived gelatinolytic enzymes, as evidenced by immunoprecipitation. In order to identify the neutrophil gelatinase, the activity in conditioned medium from human neutrophil suspensions was purified by affinity chromatography on a gelatin substrate. The 91‐kDa active enzyme was further separated from other stainable protein bands by classical SDS/PAGE and blotting to a solid support. Amino‐terminal sequence analysis of blotted proteins showed that the 91‐kDa enzyme is a truncated form of tumor‐derived 92‐kDa gelatinase (type IV collagenase), lacking eight residues at the NH2‐terminus. Sequence analysis of enzymatically inactive cleavage products of this neutrophil gelatinase demonstrated that the gelatin‐binding part of the molecule is restricted to the amino‐terminal third.Exocytosis of gelatinase‐containing granules from neutrophils occurred spontaneously within 6 h after neutrophil plating. When the cells were triggered with the phorbol ester phorbol 12‐myristate 13‐acetate, a strong secretagogue, rapid gelatinase release was observed. When granulocytes were stimulated with the neutrophil‐activating peptide interleukin‐8, maximal exocytosis occurred within 1 h. The almost immediate release of neutrophil gelatinase after stimulation of the cells with a chemotactic factor might play a key role in remodeling of the extracellular matrix during granulocyte movement in response to chemotactic stimuli.
The H+‐motive and Na+‐motive respiratory chains in Bacillus FTU subcellular vesicles
Authors: KOSTYRKO Vitaly A. , SEMEYKINA Anna L. , SKULACHEV Vladimir P. , SMIRNOVA Irina A. , VAGHINA Margarita L. , VERKHOVSKAYA Marina L. 
Published: March 2005

Respiration‐dependent pumping of Na+ and H+ into the inside‐out subcellular vesicles of alkalotolerant and halotolerant Bacillus FTU grown at alkaline pH was studied.The vesicles were shown to be competent in Na+ and H+ transport coupled to ascorbate oxidation via N,N,N′,N′‐tetramethyl‐p‐phenylenediamine or diaminodurene. The uphill Na+ uptake is strongly stimulated by either protonophores or valinomycin, whereas H+ uptake is stimulated by valinomycin and completely inhibited by protonophores. The salt of a penetrating weak base and of the penetrating weak acid, diethylammonium acetate, potentiates the stimulating effect of protonophores on Na+ uptake and abolishes H+ uptake. Na+ transport, supported by ascorbate oxidation, is resistant to 2‐heptyl‐4‐hydroxyquinoline N‐oxide, but sensitive to Ag+ and Na+ ionophore, N,N′‐dibenzyl‐N,N′‐diphenyl‐1,2‐phenylenediacetamide. Micromolar concentrations of cyanide specifically inhibit the H+ uptake but does not affect Na+ uptake. These cyanide concentrations are shown to cause 70% inhibition of respiration, complete reduction of a‐type cytochromes and partial reduction of c/b‐type cytochromes. To inhibit the remaining respiratory activity and Na+ uptake, approximately 100‐fold higher cyanide concentrations are necessary. High cyanide concentrations cause some additional increase in absorbance in the region of cytochromes c and/or b. In the presence of a high cyanide concentration, Na+ uptake can be supported by NADH oxidation by fumarate. This Na+ transport is stimulated by protonophores and diethylammonium acetate, being sensitive to very low concentrations of 2‐heptyl‐4‐hydroxyquinoline N‐oxide and Ag+. The NADH‐fumarate reductase reaction is also found to be competent in H+ uptake, which is inhibited by protonophores and by much higher 2‐heptyl‐4‐hydroxyquinoline N‐oxide concentrations, and is resistant to Ag+.It is inferred that Bacillus FTU possesses two respiratory chains: the H+‐motive and the Na+‐motive, which strongly differ in their inhibitor sensitivities. Each chain comprises at least two energy‐coupling sites which are localized in their initial and terminal segments. It has been indicated that common redox carrier(s) are present in the two chains.
Mechanism of the permissive action of dexamethasone on the glucagon‐dependent activation of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes
Authors: RUNGE Dieter , SCHMIDT Helga , CHRIST Bruno , JUNGERMANN Kurt 
Published: March 2005

Rat hepatocytes were cultured for 24 h in the presence or absence of 100 nM dexamethasone (DX). After a medium change, phosphoenolpyruvate carboxykinase (PCK) was induced by addition of glucagon at different concentrations, from physiological 0.1 nM to hyperphysiological 10 nM, again in the presence or absence of 100 nM dexamethasone.1. With dexamethasone addition during the culture and induction phase (DX+/+), 10 nM glucagon increased PCK mRNA abundance (Northern blot analysis) and activity (in vitro translation) synchronously to the same extent with maxima after 2 h and PCK enzyme activity after a time lag with a maximum after 6 h. The total detectable PCK mRNA amount was apparently also translationally active. 10 μM N6,2′‐O‐dibutyryladenosine 3′,5′‐(cyclic)phosphate (Bt2cAMP) as the second messenger had essentially the same effect as 10 nM glucagon.2. In the absence of dexamethasone during the preculture and the induction phase (DX‐/‐), 10 nM glucagon and 10 μM Bt2cAMP could enhance PCK mRNA only about half‐maximally. Glucagon or dexamethasone added alone in physiological concentrations of 0.1 nM and 100 nM, respectively, were unable to increase PCK mRNA. However, treatment of the cells with dexamethasone also enabled 0.1 nM glucagon to enhance PCK mRNA to a maximum after 2 h, independent of the presence of dexamethasone during the induction period (DX+/+ and DX+/‐ cells). Thus, dexamethasone was a permissive agent in that it shifted the sensitivity of the cells towards glucagon into the physiological concentration range.3. In the presence of dexamethasone during the culture and induction phase (DX+/+) 0.1 nM glucagon maximally enhanced the transcription of the PCK gene (nuclear run on) fourfold after 30 min; in the absence of dexamethasone during both phases (DX‐/‐) glucagon was without any effect. The overall transcriptional rate was not significantly different in cells with and without dexamethasone during the culture and induction phase (DX+/+ vs. DX‐/‐). Thus, dexamethasone acted permissively mainly on the transcription of the PCK gene.4. With culture in the presence of dexamethasone over decreasing periods of time, 1 nM glucagon could induce submaximal PCK mRNA amounts already after 1–3 h steroid culture. This restitution by dexamethasone of the PCK mRNA inducibility by glucagon was inhibited by cycloheximide. This suggested that ongoing protein synthesis was required for the permissive action of dexamethasone on the expression of the PCK gene.The results allow the following conclusions. In the presence of dexamethasone a trans‐acting protein factor was formed which allowed glucagon via cAMP to enhance the expression of the PCK gene in physiological concentrations. The dexamethasone‐induced trans‐acting factor exerted its permissive action mainly at a transcriptional level.
EPR and 1H‐NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors
Authors: MARTÍNEZ Aurora , ANDERSSON Kristoffer K. , HAAVIK Jan , FLATMARK Torgeir 
Published: March 2005

The paramagnetic iron at the active site of highly purified, catalytically active phenylalanine hydroxylase was studied by EPR at 3.6 K and one‐dimensional 1H‐NMR spectroscopy at 293 K. The EPR‐detectable iron of the bovine enzyme was found to be present as a high‐spin form (S= 5/2) in different ligand field symmetries depending on medium conditions (buffer ions) and the presence of ligands known to bind at the active site. At 3.6 K and in phosphate buffer, the paramagnetic iron is coordinated in an environment of rhombic symmetry (g= 4.3), whereas Tris buffer favours an environment of axial ligand field symmetry (g= 6.7, 5.3 and 2.0). The latter axial type of signals resembles those observed at g= 7.0, 5.2 and 1.9 for the enzyme in phosphate buffer when L‐noradrenaline is added as an active‐site ligand (inhibitor). The same proportion of iron that coordinates to L‐noradrenaline seems to be reduced by the pterin cofactor and participate in catalysis. Experimental evidence is presented that Tris inhibits the enzyme by interacting with the enzyme‐bound ferric iron and decreases its rate of reduction by the tetrahydropterin cofactor. Preincubation with dithiothreitol also inhibits the enzyme activity and prevents the reduction of its catalytically active ferric iron by pterin cofactors as well as binding of catecholamines to the enzyme. 1H‐NMR spectroscopy revealed that the substrate (l‐phenylalanine) and l‐noradrenaline bind close to the paramagnetic iron, and that the catecholamine displaces the substrate from its binding at the active site. The results support our recently proposed model for the cooperative binding of inhibitor and substrate at the active site [Martínez, A. et al. (1990) Eur. J. Biochem. 193, 211–219].
Analysis and nucleotide sequence of the genes encoding the surface‐layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis
Authors: BRÖCKL Günther , BEHR Michael , FABRY Stefan , HENSEL Reinhard , KAUDEWITZ Hubert , BIENDL Elisabeth , KÖNIG Helmut 
Published: March 2005

The genes (slgA) encoding the surface‐layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis were cloned and sequenced. The nucleotide sequences of these genes differ at only nine positions, resulting in three amino acid differences.In both organisms, the transcription start site was localized by primer extension analyses. The DNA sequence at this site conforms to the promotor box B motif for promotors of archaea. 24 nucleotides upstream of the transcription start is an A + T‐rich region, which closely resembles the consensus box A motif of promoters of methanogens. Ribosome binding sites are exactly complementary to the 3′ end of the 16S rRNA of these methanogens.Both slgA genes encode for a precursor of the mature surface‐layer protein containing 593 amino acid residues with a putative N‐terminal signal sequence of 22 amino acid residues. The deduced protein sequences contain 20 sequon structures representing possible carbohydrate‐binding sites.In comparison with other surface‐layer proteins, these obtained from the two hyperthermophilic methanogens contain unusually high amounts of isoleucine, asparagine and cysteine residues. Predicted secondary structures have a high content of β‐sheet structure (44%) and only 7%α‐helix structures.
The interaction of paramagnetic ions chelated to ATP with pyridoxal analogues
Authors: KWOK Francis , CHURCHICH Jorge E. 
Published: March 2005

Fluorescence spectroscopy, static and dynamic, was applied to deduce proximity relationships between paramagnetic metal ions chelated to ATP and the inhibitor pyridoxal oxime bound to the site of the substrate pyridoxal. The fluorescence yield of free pyridoxal oxime is considerably reduced in the presence of CoCl2 (1 mM) due to the formation of a non‐fluorescent complex.The fluorescence properties of pyridoxal oxime bound to pyridoxal kinase (50 μM, Kd= 1 μM) remain invariant in the presence of CoCl2 (1 mM) and ATP (1 mM). The results are interpreted to mean that the distances are too great for direct or first‐coordination‐sphere interaction between pyridoxaloxime and Co(II) centers.The bidentate complex Cr(III) · ATP is a competitive inhibitor of ATP (Ki= 83 μM) and its binding to pyridoxal kinase (50 μM) complexed to pyridoxal oxime (50 μM) was monitored by static and dynamic fluorescence spectroscopy. The fluorescence yield of bound pyridoxal oxime is reduced by 25% in the presence of Cr(III) · ATP (0.8 mM) leading to a decrease in fluorescence lifetime from 〈τ〉= 6.60 ns to 〈τ〉= 4.20 ns. The decrease in fluorescence yield together with a diminution in fluorescence lifetime can be explained in terms of radiationless energy transfer from excited pyridoxal oxime to Cr(III) chelated to ATP.Using Forster's equation and an efficiency of energy transfer of 0.25, it was found that a distance of 1.3 nm separates pyridoxal oxime from the Cr(III) center.