bluuebunny/crossref_metadata_2025_split huggingface dataset:
A DNA polymerase with unusual properties from the slime mold Physarum polycephalum
Authors: HOLLER Eggehard , FISCHER Heinrich , WEBER Cornelia , STOPPER Helga , STEGER Heinrich , SIMEK Helmut 
Published: March 2005

Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE‐Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA‐agarose, blue‐Sepharose. They were separated from DNA polymerase α on phosphocellulose and from each other on heparin‐Sepharose. Form HS1 enzyme was 30–40% pure and form HS2 enzyme 60% with regard to protein contents of the preparations. Form HS2 enzyme was generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60‐kDa protein associated with either a 135‐kDa (HS1) or a 110‐kDa (HS2) DNA‐polymerizing polypeptide in a 1:1 molar stoichiometry. The biochemical function of the 60‐kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as well as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis.The DNA polymerases resembled eucaryotic DNA polymerase β by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis.
Cytochrome c oxidase is three‐copper, two‐heme‐A protein
Authors: STEFFENS Guy C. M. , BIEWALD Rosemarie , BUSE Gerhard 
Published: March 2005

Metal contents of preparations of procaryotic (Paracoccus denitrificans) and eucaryotic (beef heart) cytochome c oxidases have been determined by inductively coupled plasma atomic emission spectroscopy and shown to be stoichiometrically related to the protein contents. The results show that oxidases which possess subunits I and II have three copper atoms besides hemes a and a3 (Paracoccus denitrificans, Cu: 2.97 ± 0.08 and Fe: 2.09 ± 0.10; bovine heart, Cu: 2.83 ± 0.07 and Fe: 1.94 ± 0.12). Together with data reported for the c1aa3 oxidase from Thermus thermophilus, the following conclusions can be drawn. Subunit I binds two copper atoms and both hemes a and a3 and thus is the universal terminal oxidase of this spectral type. Subunit II binds one copper and functions as an electron conductor.The mitochondrial respiratory complex IV binds, in addition to three copper and two hemes a, stoichiometric amounts of magnesium and zinc (bovine heart Mg: 0.98 ± 0.05 and Zn: 1.01 ± 0.04).
Chemical, immunological and biological properties of peptides like vasoactive‐intestinal‐peptide and peptide‐histidine‐isoleucinamide extracted from the venom of two lizards (Heloderma horridum and Heloderma suspectum)
Authors: VANDERMEEERS Andé , GOURLET Philippe , VANDERMEERS‐PIRET Marie‐Claire , CAUVIN Annick , DE NEEF Philippe , RATHE Jean , SVOBODA Michal , ROBBERECHT Patrock , CHRISTOPHE Jean 
Published: March 2005

1. Having previously isolated helodermin, the major peptide like vasoactive‐intestinal‐peptide and peptide‐histidine‐isoleucinamidc, from the venom of the lizard Heloderma suspectum, we decided on a systematic exploration of all (VIP‐PHI)‐like peptides present in the venom of another lizard of the Helodermatidae family: Heloderma horridum.2. Six (VIP‐PHI)‐like peptides (PHH1 to 6) were purified to homogeneity from the venom of the lizard H. horridum with PHH3 and PHH4 representing two minor forms. All peptides cross‐reacted in radio‐immunoassays for helodermin and PH1 but not for VIP. They yielded four fragments (T1 to T4) after trypsin digestion. T1, T2 and T3 showed the same retention time by reverse‐phase HPLC and the same amino acid composition; the differences were confined to T4, the C‐terminal sequence. PHH5 and PHH6 were found to be identical to synthetic helospectins I and II respectively. PHH1 and PHH3 probably resulted from a secondary modification of PHH5, while PHH2 and PHH4 derived from PHH6. Thus, the VIP‐like peptides, previously called helospectins, are in fact typical of H. horridum venom.3. We confirmed that helodermin is the major (VIP‐PHI)‐like peptide of the venom of H.suspectum and observed its absence in H. horridum venom. Also, we found that positions 8 and 9 of helodermin are occupied by two Glu residues instead of two Gln as previously published. Helospectin‐like material was also present in H. suspectum venom but in very small amount.4. In both venom all VIP‐like peptides were equally potent and efficient when tested for (a) their ability to occupy VIP as well as secretin receptors in rat pancreatic membranes and VIP receptors in rat liver membranes, and (b) the ensuing activation of adenylate cyclase in both membrane preparations.
Use of a polymer‐bound flavin derivative for the rapid regeneration of NAD(P)+ from NAD(P)H in dehydrogenase systems
Authors: MONTAINE France , LENDERS Jean‐Pierre , CRICHTON Robert R. 
Published: March 2005

An aldehyde derivative of riboflavin was covalently attached by reductive alkylation to soluble polycationic supports. The flavopolymers so obtained were stable under operational conditions. The catalytic efficiency towards oxidation of NADH by these flavopolymers was demonstrated, and the kinetic parameters (Km and Kcat) revealed an overall catalytic efficiency (Kcat/Km) 185‐fold greater compared to riboflavin.Various factors affecting the chemical regeneration of NAD+ from NADH such as pH, ionic strength, nature of the buffer etc. were studied. The most interesting result was the highly favourable influence of borate ions which increased the reaction rate by a factor 2–4 compared to the other buffers.The flavopolymers are very effective for in situ recycling of NAD(P)+. With up to 300‐fold NADH → NAD+ conversions for the system using yeast alcohol dehydrogenase and up to 1500‐fold NADPH → NADP+ regenerations for the system using glucose‐6‐phosphate dehydrogenase. These flavopolymers are superior to previous chemical recycling systems.
Desmosine and isodesmosine as cross‐links in the hinge‐ligament protein of bivalves
Authors: KIKUCHI Yasuo , TSUCHIKURA Osamu , HIRAMA Masahiro , TAMIYA Nobuo 
Published: March 2005

1. Desmosine and isodesmosine were detected in an invertebrate molluscan species, i.e. in an insoluble protein in the hinge ligament of a bivalve species, Sakhalin surf clam (Pseudocardium sachalinensis, in family Mactridae). The protein is rich in glycine and methionine S‐oxide but devoid of hydroxyproline and hydroxylysine.2. 3,3′‐Methylenebistyrosine was also detected in the HCl hydrolysate of the hinge‐ligament protein, but it was found to be an artefact produced from tyrosine and formaldehyde derived from methionine S‐oxide during the HCl hydrolysis of the protein.
Differentiation of the drug‐binding sites of calmodulin
Authors: ZIMMER Manfred , HOFMANN Franz 
Published: March 2005

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various ‘calmodulin antagonists’ for these sites was investigated. The Ki values for the inhibition of calmodulin‐activated cyclic‐nucleotide phosphodiesterase and myosin light‐chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four groups. Group I and II compounds inhibited competitively the activation of the phosphodiesterase and myosin light‐chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light‐chain kinase at identical concentrations. In contrast, group II compounds inhibited the activation of the phosphodiesterase at 5–10‐fold lower concentrations than that of myosin light‐chain kinase. Group III compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 μM and did not affect the activation of myosin light‐chain kinase.Binding of [3H]bepridil to calmodulin under equilibrium conditions yielded one high‐affinity site (apparent Kd 0.4 μM) and four low affinity sites (apparent Kd 44 μM). Group I compounds interfered with the binding of bepridil to the high and low‐affinity sites in a competitive manner. Group II compounds interfered in a noncompetitive manner with the high‐affinity site and apparently competed only with one of the low‐affinity sites. Group III compounds did not compete with any of the bepridil‐binding sites. Nimodipine, a group III compound, bound to one site on calmodulin with a Kd value of 1.1 μM. Other dihydropyridines competed with [3H]nimodipine for this site. The group I and II compounds, trifluoperazine and prenylamine, did not affect the binding of [3H]nimodipine.These data show that ‘calmodulin antagonists’ can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes.
Rapid purification of protein kinase C from rat brain
Authors: WOOTEN Marie W. , VANDENPLAS Michel , NEL André E. 
Published: March 2005

We describe a rapid purification of protein kinase C from rat brain cytosol employing a specific substrate, protamine‐coupled to agarose. Sequential chromatography on DEAE‐Sephacel, phenyl‐Sepharose CL‐4B, and protamine‐agarose columns resulted in a 1500‐fold purification of protein kinase C. SDS‐PAGE analysis of the purified enzyme resolved a doublet protein of 77–80 kDa. This doublet was recognized by a polyclonal antiserum against protein kinase C. Proteolytic digestion of each protein band generated similar peptide fragments.The underlying principle of the protamine sulfate purification method was also clarified. Protamine can serve as a Ca2+/phospholipid‐independent substrate. We demonstrate phosphorylation of protamine on the column: phosphorylated protamine did not bind the enzyme with the same affinity and this covalent modification was most probably responsible for releasing the bound enzyme from the column after addition of Mg2+ and ATP. The C kinase inhibitor, H7, inhibits protamine phosphorylation in a dose‐dependent fashion but does not prevent binding of the enzyme to a protamine‐agarose column. We therefore conclude that protamine interacts with the active center of the enzyme enabling it to be phosphorylated, upon which it loses its binding affinity for C kinase.
The calcium channel antagonists receptor from rabbit skeletal muscle
Authors: BARHANIN Jacques , COPPOLA Thierry , SCHMID Annie , BORSOTTO Marc , LAZDUNSKI Michel 
Published: March 2005

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200–110, (−)[3H]D888 and d‐cis‐[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1.Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170000 under non‐reducing conditions and of two polypeptides of Mr 140000 and 32000 under disulfide‐reducing conditions. Iodination of the protein of Mr 170000 and immunoblots experiments with antisera directed against the differents components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170000 composed of a polypeptide chain of Mr 140000 associated to one polypeptide chain of Mr 32000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29000 and 25000. Peptide mapping of these polypeptide chains and analysis of their cross‐reactivity with sera directed against the proteins of Mr 170000 and 32000 demonstrated that they were degradative products of the Mr 32000 component.Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20–22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140‐kDa and 32‐kDa components originate from a single polypeptide chain of Mr 170000 which is cleaved by a proteolysis upon purification is discussed.
Cloning and characterization of the pyrF operon of Salmonella typhimurium
Authors: THEISEN Michael , KELLN Rod A. , NEUHARD Jan 
Published: March 2005

The pyrF gene of Salmonella typhimurium encoding the sixth enzyme of pyrimidine nucleotide biosynthesis, OMP decarboxylase, was isolated from a pyrF‐complementing R' factor. A 2.0‐kbp DNA fragment, generated by PvuI cleavage, was subsequently subcloned into the multicopy vector pBR322 and shown to contain the intact pyrF gene. Bacterial strains harbouring the resulting plasmid contain 15–20‐fold elevated levels of OMP decarboxylase, and these levels increase 4–5‐fold during uracil starvation.Experiments utilizing minicells identified the gene product as a polypeptide with a molecular mass of approximately 27 kDa. Furthermore, it was found that the pyrF gene is expressed as the first gene of a bicistronic operon, wherein the second gene encodes an 11‐kDa polypeptide of unknown functions. The complete nucleotide sequence of the pyrF operon was determined. An open reading frame, encoding a polypeptide with a calculated molecular mass of 26213 Da, was deduced to be the coding region for pyrF. Another open reading frame, with a translational start codon which overlaps the translational stop codons of the pyrF gene, encodes a polypeptide of 11513 Da. This open reading frame represents the coding region for the second gene of the operon, orfF. S1‐nuclease mapping indicated that pyrF transcription is initiated 54 bases upstream of the translational start. The leader region does not show any features resembling the attenuators found preceding the pyrBI operon and the pyrE gene.
Purification and characterization of extracellular α‐amylase and glucoamylase from the yeast Candida antarctica CBS 6678
Authors: DE MOT René , VERACHTERT Hubert 
Published: March 2005

An α‐amylase and a glucoamylase were purified to homogeneity from the culture fluid of β‐cyclodextringrown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G‐75 sf, Ultrogel AcA 54), DEAE‐Sephacel chromatography, hydroxyapatite chromatography and affinity chomatography on acarbose—AH‐Sepharose 4B.Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparent relative molecular mass, sedimentation coefficient (S°20, W), isoelectric point, absorption coefficient (280 nm), pH and temperature optima were estimated as 48 500, 4.7 S, 10.1, 1.74 cm2 mg−1, 4.2 and 57°C, respectively, for glucoamylase and as 50 000, 4.9 S, 10.3, 1.53 cm2 mg−1, 4.2 and 62°C, respectively, for α‐amylase.Kinetic analyses indicated that both enzymes preferentially hydrolyzed high‐molecular‐mass substrates, including some raw starches. α‐Amylase was active on cyclodextrins, whereas debranching activity was demonstrated for glucoamylase. Trestatins were potent inhibitors of both α‐amylase (Ki < 1 μM) and glucoamylase (Ki < 0.1 μM), being more effective than Bay e 4609 (Ki < 10 μM). Glucoamylase was selectivity and strongly inhibited by acarbose (Ki < 0.1 μM). Activity of the latter enzyme was also affected by 1‐deoxynojirimycin (Ki < 1 mM), maltitol and amino alcohols (Ki < 10 mM).Unlike α‐amylase, glucoamylase adsorbed strongly onto raw starch, the adsorption site being non‐identical with the active site.
A simple approach to detect active‐site‐directed enzyme‐enzyme interactions
Authors: VÉRTESSY Beáta , OVÁDI Judit 
Published: March 2005

A novel approach has been elaborated to identify the mechanism of intermediate transfer in interacting enzyme systems. The aldolase/glycerol‐3‐phosphate‐dehydrogenase enzyme system was investigated since complex formation between these two enzymes had been demonstrated.The kinetics of dihydroxyacetone phosphate conversion catalyzed by the dehydrogenase in the absence and presence of aldolase was analyzed. It was found that the second‐order rate constant (kcat/Km) of the enzymatic reaction decreases due to the formation of a heterologous complex. The decrease could be attributed to an increase of the Km value since kcat did not change in the presence of aldolase. In contrast, an apparent increase in the second‐order rate constant of dihydroxyacetone phosphate conversion by the dehydrogenase was observed if the triose phosphate was produced by aldolase from fructose 1,6‐bisphosphate (consecutive reaction). Moreover, no effect of dihydroxyacetone phosphate on the dissociation constant of the heterologous enzyme complex could be detected by physico‐chemical methods.The results suggest that the endogenous dihydroxyacetone phosphate produced by aldolase complexed with dehydrogenase is more accessible for the dehydrogenase than the exogenous one, the binding of which is impeded due to steric hindrance by bound aldolase.
Solubilization and characterization of guinea‐pig pancreatic somatostatin receptors
Authors: ZEGGARI Mustafa , VIGUERIE Nathalie , SUSINI Christiane , GARNIER Martine , ESTEVE Jean‐Pierre , RIBET André 
Published: March 2005

The solubilization of somatostatin receptors from guinea‐pig pancreas by different non‐denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I‐[Tyr11]somatostatin or its analogue 125I‐[Leu8, DTrp22, Tyr25]somatostatin 28, to pancreatic plasma membranes.The somatostatin‐receptor complexes were solubilized in a high yield by Zwittergent 3–14 (3‐[tetradecyldimethylammonio]‐1‐propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X‐100, Chaps (3‐[cholamidopropyldimethylammonio]‐1‐propanesulfonate) and octyl β‐D‐glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol.In order to characterize solubilized somatostatin‐receptor comples, membranes receptors were covalently labelled using N‐5‐azido‐2‐nitrobenzoyloxysuccinimide as cross‐linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr= 93000 which interacted with the two radioligands.In addition, a similar component of Mr= 88000 was characterized after analysis by SDS‐PAGE of membrane receptors covalently cross‐linked with 125I‐[Leu8, DTrp22, Tyr25]somatostatin 28 by different heterobifunctional reagents: N‐5‐azido‐2‐nitrobenzoyloxysuccinimide, N‐hydroxysuccinimidyl 4‐azidobenzoate, N‐succinimidyl 6‐(4′‐azido‐2′‐nitrophenylamino)hexanoate. Optimal cross‐linking results were obtained with N‐5‐azido‐2‐nitrobenzoloxysuccinimide.The solubilized somatostatin‐receptor complex was adsorbed to wheat‐germ agglutinin‐agarose column and eluted by specific sugars.We concluded that the guinea‐pig pancreatic somatostatin receptor in the membrane and in the non‐denaturing detergent solution behaves as a protein monomer of apparent Mr∼ 85000–90000. The somatostatin receptor is a glycoprotein which contains complex‐type carbohydrate chains.
Apparent unmasking of [3H]GDP binding in rat brown‐fat mitochondria is due to mitochondrial swelling
Authors: NEDERGAARD Jan , CANNON Barbara 
Published: March 2005

1. The presence of and biochemical background for the so‐called ‘unmasking’ phenomenon in rat brown‐fat mitochondria was investigated (i.e. the apparent increase in [3H]GDP binding to the ‘uncoupling’ protein thermogenin, without a concomitant increase in the amount of the protein).2. It was found that an unmasking could be observed both 1 h after norepinephrine injection and after 1 h cold stress, provided that the rats were preacclimated to 28°C. The unmasking could be observed both when a filtration method and when a centrifugation method for determination of [3H]GDP‐binding capacity were used; however, the absolute values were higher with the filtration method.3. Based on observations of slower cytochrome‐c oxidase sedimentation during centrifugation, the possibility that the matrix volume of brown‐fat mitochondria isolated from warm‐acclimated animals was smaller than that of cold‐stressed animals was investigated with 3H2O. The cold stress increased the matrix volume from being nearly non‐existent to about 1 μl/mg. A preswelling procedure in an ionic medium could similarly increase the matrix volume in mitochondria from warm‐acclimated animals but was without significant effect in the already swollen mitochondria from cold‐stressed animals or from animals adapted to a lower temperature.4. In mitochondria from warm‐acclimated animals, the ionic preswelling procedure was fully able to increase the apparent amount of GDP binding to that observed in mitochondria from cold‐stressed animals, but it was practically without effect on GDP binding in mitochondria from cold‐stressed animals or from animals acclimated to a lower temperature.5. It is concluded that the apparent ‘unmasking’ phenomenon, observed when the tissue is less activated than in normal control situations, is not (as hitherto anticipated) due to a specific change in thermogenin as such, but is a reflection of a general mitochondrial phenomenon.
Control of uncoupling protein in brown‐fat mitochondria by purine nucleotides
Authors: KOPECKÝ Jan , JEŽEK Petr , DRAHOTA Zdeněk , HOUŠTĚK Josef 
Published: March 2005

The uncoupling protein (UP) of isolated brown adipose tissue mitochondria was studied with respect to the mechanism of control of UP function by purine nucleotides. Passive transport of H+ and Cl− was followed simultaneously in a KCl medium. With both GDP and ATP a higher sensitivity of Cl− transport (apparent Ki= 2.2 μM and 4.7 μM respectively) than of H+ transport (apparent Ki= 7.7 μM and 34 μM respectively) was observed. Chemical modification of isolated mitochondria by diazobenzenesulfonate (DA BS) up to 75 μmol/mg protein did not affect the transport, its ionic selectivity and regulation by endogenous free fatty acids. In contrast, the sensitivity to purine nucleotides of both H+ and Cl− translocation was decreased (apparent Ki increased 71 and 47 times respectively). DABS decreased the affinity of [3H]GDP for the specific nucleotide‐binding site on mitochondria (Kd increased from 2.7 μM to 13 μM) and depressed, to a smaller extent, the GDP‐binding capacity. Correlation between occupancy of the specific nucleotide‐binding site by GDP and inhibition of transport yielded a linear relationship for Cl− transport in control mitochondria, a biphasic correlation was obtained.The results show that different structural parts of UP are involved in transport and its control by the regulatory ligands and that, in addition to binding of purine nucleotides to UP, the inhibition of ion transport by purine nucleotides depends on an intrinsic factor modulating the inhibitory effect.