bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica
Authors: SAKAI Michizo , SAISU Hideo , ABE Teruo 
Published: March 2005

The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared.

Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj‐601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj‐601‐Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8.
The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme form N. japonica contained more A8 than A12, while that of T. californica contained more A12.
After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen‐like tails.
On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140000, 100000, 70000 and 60000, while that from T. californica gave bands of Mr 140000, 100000, 70000 and 55000.
The bands of Mr 70000 and 140000 were monomers and non‐reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60000 and 55000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components.
The Mr 100000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6–8% of the two preparations.
The catalytic subunits and the Mr 100000 subunits bound concanavalin A, indicating that they are glycoproteins.
The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen‐like tails.
The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity.
A nuclear‐magnetic‐resonance study of the binding of novel N‐hydroxybenzenesulphonamide carbonic anhydrase inhibitors to native and cadmium‐111‐substituted carbonic anhydrase
Authors: BLACKBURN G. Michael , MANN Brain E. , TAYLOR Brain F. , WORRALL Andrew F. 
Published: March 2005

Various ring‐ and nitrogen‐substituted benzenesulphonamides have been prepared and tested as potential inhibitors of carbonic anhydrase. N‐Methoxysulphonamides showed no inhibitory activity, as predicted by the classic work of Krebs on N‐substituted inhibitors. By contrast, N‐hydroxysulphonamides proved to be very effective inhibitors of carbonic anhydrase.Using 111Cd‐NMR it has been possible to analyse the molecular interaction of 4‐fluoro‐N‐hydroxybenzene‐sulphon[15N]amide, with 111Cd‐substituted bovine carbonic anhydrase. A large cadmium‐111: nitrogen‐15 spin‐coupling shows that this inhibitor is directly bound to the metal via its nitrogen rather than through an oxygen atom. The mode of this binding is similar to that for the unsubstituted sulphonamide inhibitor, 4‐fluorbenzenesulphon[15N]amide.The 111Cd‐chemical shift of the signal for the inhibited enzyme shows that the N‐hydroxysulphonamide is bound as its anion. Form the relative intensities of free and complexed enzyme signals it can be deduced that the cadmium enzyme complex with the N‐hydroxysulphonamide has a longer life‐time than that formed with the unsubstituted sulphonamide. By contrast, native zinc‐containing bovine carbonic anhydrase shows similar I50 values with both of these sulphonamides. Attempts to monitor the binding using 15N‐NMR were unsuccessful, possible due to a very long relaxation time for the nitrogen nucleus in the N‐hydroxysulphonamide when found to the enzyme leading to loss of the 15N signal.
Analysis of protein A encoded by a mutated gene of Staphylococcus aureus Cowan I
Authors: GUSS Bengt , LEANDER Karen , HELLMAN Ulf , UHLÉN Mathias , SJÖQUIST John , LINDBERG Martin 
Published: March 2005

The protein A (spa) genes from Staphylococcus aureus Cowan I and a mutant strain of Cowan I called V‐1 earlier suggested to produce a monovalent IgG‐binding protein A have been cloned in Escherichia coli. The DNA sequences coding for the IgG‐binding part of the spa genes from both strains have been determined and compared with each other and with a partial amino acid sequence of purified protein A from strain V‐1.The nucleotide sequence of the spa gene from strain V‐1 reveals an NH2‐terminally located IgG‐binding region homologous to region E first reported for strain 8325‐4, region D and the major portion of region A. The amino acid sequence analysis of the purified protein A from this strain also shows the presence of regions E and D but only a minor part of region A. Reversed‐phase high‐performance liquid chromatography fractionation of purified protein A from strain V‐1 revealed that the preparation was heterogenous, containing mainly two peptides with different abilities to bind IgG molecules.A shuttle vector containing the cloned protein A gene from V‐1 was constructed and transformed into different strains of S. aureus and the produced protein A was purified and analysed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis.
Proteinase inhibitors and dendrotoxins
Authors: DUFTON Mark J. 
Published: March 2005

The amino acid sequences of four presynaptically active toxins from mamba snake venom (termed ‘dendrotoxins’) were compared systematically with homologous sequences of members of the proteinase inhibitor family (Kunitz). A comparison based on the complete sequences revealed that relatively few amino acid changes are necessary to abolish antiprotease activity and convert a proteinase inhibitor into a dendrotoxin. When comparison centred only on the sequence segments known to comprise the antiprotease site of bovine pancreatic trypsin inhibitor, the dendrotoxins were clearly classified apart from all the known inhibitors. Since the mode of action of the bovine pancreatic trypsin/kallikrein inhibitor involves β sheet formation with the enzyme, predictions were obtained for this secondary structure in the region of the ‘antiprotease site’ throughout the homologues. Again, the dendrotoxins were clearly distinguished from the inhibitors.Structure/activity analyses, based on the crystal structures of inhibitor/enzyme complexes, suggest that unlike proteinase inhibitors, dendrotoxins might specifically co‐ordinate the active‐site ‘catalytic’ histidine residues of serine proteases. Although the significance of this remains to be studied, the presynaptic target is expected to involve an as yet uncharacterised member of the serine protease family.
Assignment of conserved amino acid residues to the ATP site in the protein kinase domain of the receptor for epidermal growth factor
Authors: VOGEL Sabine , FREIST Wolfgang , HOPPE Jürgen 
Published: March 2005

The ATP substrate site in the epidermal growth factor (EGF) receptor was mapped by using a series of 26 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [γ‐32p]ATP.The enzyme seems to interact specifically with the β‐phosphate in an iron‐pair bond and with the N‐6 amino group at the adenine in a hydrogen bond. With ribosyl‐2‐aminopurine triphosphate and GTP, the enzyme most likely recognizes the 2‐amino group in a hydrogen bond. This high specificity for ATP and GTP is unique for the ATP site in the EGF receptor among all investigated protein kinases.The available data on the interaction between ATP derivatives and protein kinases were used to assign conserved amino acid residues found in diverse protein kinases to the ATP site in this type of enzyme.
DNA‐binding domains of human plasma fibronectin
Authors: SIRI Annalisa , BALZA Enrica , CARNEMOLLA Barbara , CASTELLANI Patrizia , BORSI Laura , ZARDI Luciano 
Published: March 2005

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA‐cellulose and heparin‐Sepharose affinity chromatography columns was performed by NaC1 linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaC1 concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA‐cellulose or heparin‐Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaC1, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA‐cellulose at 280 mM NaC1 and from the heparin‐Sepharose column at 210 mM.Furthermore, we have studied the interaction of thermolysin‐digested fibronectin both with DNA‐cellulose and heparin‐Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.
Ion fluxes changes during early stages of Schistosoma manson
Authors: TARRAB‐HAZDAI Rebeca , SAGI‐EISENBERG Ronit , BRENNER Varda , ARNON Ruth 
Published: March 2005

The relationship between the average membrane potential (ΔΨav) and sensitivity to complement action of the Schistosoma mansoni parasite was explored. The average membrane potential was estimated by measuring the uptake of [3H]tetraphenyl phosphonium ([3H]Ph4P+). The parasites take up Ph4P+ indicating the existence of a negative internal plasma potential which is in part dependent on the transmembrane K+ gradient, maintained by an active Na+/K+‐ATPase. Values for Ph4P+ uptake could be corrected for mitochondrial accumulation by employing the protonophore carbonylcyanide m‐chlorophenylhadrazone (CCCP), which collapses the mito‐chondrial potential. The plasma membrane potential derived by this technique was in the range of ‐60 mV.Transformation of this parasite, from its early cercaria stage to the adult worm, was associated with changes in the average membrane potential. The apparent hyperpolarization, which accompanies transformation, may be related to changes in ionic permeability and morphology which occur concomitantly.Complement acting through both the classical and alternative pathways was found to affect the potential of the parasite in its early developmental stages. The correlation between effects on ΔΨav and sensitivity to complement action, indicates that the complement‐induced changes in ΔΨav are indeed tightly associated with its mode of action. Treatment of the parasite with complement resulted in net hyperpolarization of the membrane indicating that hyperpolarization rather than depolarization of the membrane is linked to the primary non‐lethal action of complement.
Functional incorporation of beef‐heart cytochrome c oxidase into membranes of Streptococcus cremoris
Authors: DRIESSEN Arnold J. M. , de VRIJ Wim , KONINGS Wil N. 
Published: March 2005

Beef heart mitochondrial cytochrome c oxidase has been incorporated into membrane vesicles derived from the homofermentative lactic acid bacterium Streptococcus cremoris. Proteoliposomes containing cytochrome c oxidase were fused with the bacterial membrane vesicles by means of a freeze/thaw sonication technique. Evidence that membrane fusion has taken place is presented by the demonstration that nonexchangeable fluorescent phospholipid probes, originally present only in the bacterial membrane or only in the liposomal membrane, are diluted in the membrane after fusion and, by sucrose gradient centrifugation, indicating a buoyant density of the membranes after fusion in between those of the starting membrane preparations. The fused membranes are endowed with a relatively low ion permeability which makes it possible to generate a high proton motive force (100 mV, inside negative and alkaline) by cytochrome‐c‐oxidase‐mediated oxidation of the electron donor system ascorbate/N,N,N′,N′‐tetramethyl‐p‐phenylenediamine/cytochrome c. In the fused membranes this proton motive force can drive the uptake of several amino acids via secondary transport systems.The incorporation procedure described for primary proton pumps in biological membranes opens attractive possibilities for studies of proton‐motive‐force‐dependent processes in isolated membrane vesicles from bacterial or eukaryotic origin which lack a suitable proton‐motive‐force‐generating system.
The implication of a plastid‐derived factor in the transcriptional control of nuclear genes encoding the light‐harvesting chlorophyll a/b protein
Authors: BATSCHAUER Alfred , MÖSINGER Egon , KREUZ Klaus , DÖRR Inge , APEL Klaus 
Published: March 2005

In carotenoid‐deficient albina mutants of barley and in barley plants treated with the herbicide Norflurazon the light‐dependent accumulation of the mRNA for the light‐harvesting chlorophyll a/b protein (LHCP) is blocked. Thus, the elimination of a functional chloroplast, either as a result of mutation or as a result of herbicide treatment, can lead to the specific suppression of the expression of a nuclear gene encoding a plastid‐localized protein. These results confirm and extend earlier observations on maize [Mayfield and Taylor (1984) Eur. J. Biochem. 144, 79–84]. The inhibition of mRNA accumulation appears to be specific for the LHCP; the mRNAs encoding the small subunit of ribulose‐1,5‐bisphosphate carboxylase and the NADPH:protochlorophyllide oxidoreductase are relatively unaffected. The failure of the albina mutants and of Norflurazon‐treated plants to accumulate the LHCP mRNA is not exclusively caused by an instability of the transcript but rather by the inability of the plants to enhance the rate of transcription of the LHCP genes during illumination. Several chlorophyll‐deficient xantha mutants of barley, which are blocked after protoporphyrin IX or Mg‐protoporphyrin, and the chlorophyll‐b‐less mutant chlorina f2 accumulate the LHCP mRNA to almost normal levels during illumination. Thus, if any of the reactions leading to chlorophyll formation is involved in the control of LHCP mRNA accumulation it should be one between the formation of protochlorophyllide and the esterification of chlorophyllide a.While the nature of the regulatory factor(s) has not been identified our results suggest that, in addition to phytochrome (Pfr), plastid‐dependent factors are required for a continuous light‐dependent transcription of nuclear genes encoding the LHCP.
The phosphate group of 3‐phosphoglycerate accounts for conformational changes occurring on binding to 3‐phosphoglycerate kinase
Authors: TOMPA Péter , HONG Pham Thuy , VAS Mária 
Published: March 2005

Steady‐state kinetic study of the inhibition of 3‐phosphoglycerate kinase reaction by the substrate analogues d‐glycerol 3‐phosphate, 2‐phosphoglycolate, tartronate and malonate revealed competition with respect to 3‐phosphoglycerate. d‐Glycerate had no detectable inhibitory effect. The data indicate that (a) the phosphate of 3‐phosphoglycerate plays an essential role in the formation of its complex with the enzyme and, taking into account the relatively strong binding of 3‐phosphoglycerate, (b) the two charged groups of the substrate might cause a synergic interaction with the protein. The carboxyl‐lacking d‐glycerol 3‐phosphate is a non‐competitive inhibitor with respect to MgATP, while all the investigated carboxyl‐containing inhibitors compete for MgATP binding.The inhibitory analogues of 3‐phosphoglycerate reduce the reactivity of both the two fast‐reacting and the five slow‐reacting thiol groups of the enzyme molecule. In the case of the fast‐reacting thiols the effect is specifically associated with the presence of a ligand's phosphate group. Similarly mainly the phosphate‐containing nucleotides and analogues slow down significantly the reaction rate of the fast‐reacting thiols, while adenosine is less effective and the competitive inhibitor adenine has no effect at all. MgADP has an especially dramatic effect as compared to MgATP, in line with the known X‐ray structural data.The fast‐reacting thiols are of particular interest, since their reactivity is possibly controlled by ligand‐induced conformational changes. This is shown by the similar ligand protection against alkylation irrespective of the reagent's electrostatic charge (iodoacetamide or iodoacetate) and also by the similar substrate‐binding properties of carboxamidomethylated and the unmodified enzyme.
The phosphoenolpyruvate‐dependent fructose‐specific phosphotransferase system in Rhodopseudomonas sphaeroides
Authors: LOLKEMA Juke S. , TEN HOEVE‐DUURKENS Ria H. , ROBILLARD George T. 
Published: March 2005

Two interrelated sites have been detected on the fructose carrier in Rhodopseudomonas sphaeroides: an activity‐linked dithiol and a Zn2+‐binding site. Binding of Zn2+ brings EFruII into a new conformation that to some extent mimics the conformation of phosphorylated EFruII, an essential intermediate in the turnover of the enzyme. Binding of zinc to EFruII or phosphorylating the enzyme protects it against trypsin inactivation relative to the dephos‐phorylated zinc‐free enzyme.A dithiol is essential for activity. Interchanges between the redox states of the enzyme can be brought about by dithiothreitol and ferricyanide, but not, or very slowly, by molecular oxygen. The dithiol is protected, in the EFruII‐Zn2+ complex, against (a) alkylation by MalNEt, (b) reversible oxidation by Fe(CN)3‐6 and Cu2+, (c) irreversible oxidation by Cu2+. The pK value of the activity linked thiol is 7.8. Protection experiments show that the dithiol is not located in any of the substrate‐binding sites. The redox state of the enzyme does not influence the rate of inactivation of EFruII by trypsin.
Subcellular localization of transferrin protein and iron in the perfused rat liver
Authors: SIBILLE Jean‐Claude , OCTAVE Jean‐Noël , SCHNEIDER Yves‐Jacques , TROUET André , CRICHTON Robert 
Published: March 2005

The subcellular localization of 3H‐labelled 59Fe‐loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques.More than 96% of the 59Fe present in the liver of rats perfused with 59Fe‐labelled transferrin is recovered in the parenchymal cells.Rat livers were perfused with 10 μg/ml 3H‐labelled 59Fe‐saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant (S) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients. 3H label distributes essentially around densities of 1.13–1.14 g/ml overlapping to a large extent with the distribution of galactosyltransferase, the marker enzyme of the Golgi complex.However, after treatment with low concentrations of digitonin the 3H. label dissociates from galactosyltransferase and is shifted to higher densities, suggesting an association of transferrin with cholesterolrich endocytic vesicles which could derive from the plasma membrane. 59Fe is mostly found in the supernatant fraction largely in the form of ferritin, as indicated by its reaction with antiferritin antibodies. In the mitochondrial fraction the density distribution of 59Fe suggests an association with lysosomes and/or mitochondria. In contrast to the lysosomal enzyme cathepsin B, the density distribution of 59Fe was only slightly affected by pretreatment of the rats with Triton WR 1339, suggesting its association with the mitochondria, At 15°C, 59Fe and 3H labels are recovered together in low‐density endocytic vesicles.On the basis of our results we suggest that, at low extracellular transferrin concentration, iron uptake by the liver involves endocytosis of the transferrin protein. The complex is interiorized in low‐density acidic vesicles where iron is released. The iron passes into the cytosol, where it is incorporated into ferritin and into the mitochondria. The iron‐depleted transferrin molecule would then be returned to the extracellular medium during the recycling of the plasma membrane.
Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells
Authors: MOUSTACAS Anne‐Marie , NARI Johanès , DIAMANTIDIS Grégoire , NOAT Georges , CRASNIER Martine , BOREL Maguy , RICARD Jacques 
Published: March 2005

The pectin methyl esterase from soybean cell walls has been isolated and purified to homogeneity. It is a protein with a relative molecular mass close to 33 000. The enzyme is maximally active at a pH close to 8 and its pH dependence may be explained by a classical Dixon model, where the two interconvertible enzyme ionization states coexist.The outflux of protons from cell walls, upon raising the ionic strength, may be taken as an indirect estimate of the fixed charge density. If the cell‐wall fragments are pre‐incubated at pH values between 5 and 9, the outflux of protons rises with the pH of pre‐incubation. This implies, as postualted from the theory developed in the preceding paper, that alkaline pH favours the activity of pectin methyl esterase and that this enzyme effectively generates the fixed negative charges of the cell wall. Therefore the pectin methyl esterase reaction builds up the Donnan potential, ΔΨ, at the cell surface.The cell‐wall charge density, estimated from the proton outflux, as well as from the titration of methyl groups on the cell wall, reaches a maximum between the third and the fourth day of growth. While the cell‐wall volume increases and reaches a plateau, the fixed charge density increases at first and then declines. This is understandable if one assumes that the building up of a high charge density is a co‐operative phenomenon and that the local pH inside the wall rises during cell growth. When both the cell‐wall volume and the charge density increase together, this suggests that the local pH inside the wall lies within the critical pH range associated with the steep response of the system. When the cell‐wall volume increases together with a decrease of the fixed charge density, the local pH should have dropped below this critical pH range. Under these conditions the pectin methyl esterase remains inactive, or poorly active.As the number of fixed negative charges increases, calcium becomes tightly bound to cell walls. This binding is so tight that the net charge density is minimum when the calcium concentration is maximum.The experimental results, presented above, offer experimental support to two important ideas discussed in the preceding paper, namely that pectin methyl esterase reaction builds up the Donnan potential at the cell surface, and that this response may be co‐operative with respect to pH.
Removal of ferritin‐bound iron by DL‐dihydrolipoate and DL‐dihydrolipoamide
Authors: BONOMI Franco , PAGANI Silvia 
Published: March 2005

The naturally occurring dithiols DL‐dihydrolipoate and DL‐dihydrolipoamide were tested for their ability in the removal of ferritin‐bound iron. Both compounds remove the iron stored inside the protein by complexing it in the ferric form. The iron can be reduced to the ferrous form by excess dithiol, but this is not necessary for complete removal. Reaction is complete in few hours and, at molar ratios of chelator to metal higher than 10, more than 60% of the ferritin‐bound iron was removed. The amount of iron stored in the ferritin molecule does not affect the rate and the yield of the removal reaction.The iron‐removing ability of DL‐dihydrolipoate was found to be identical to that of an equimolar solution of sodium dithionite, and to be pH‐dependent.Results are discussed in terms of the molecular architecture of ferritin and of the chelators, and their possible physiological relevance is pointed out.