bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrous oxide reductase from denitrifying
Authors: COYLE Catherine L. , ZUMFT Walter G. , KRONECK Peter M. H. , KÖRNER Heinz , JAKOB Wolfgang
Published: March 2005

Nitrous oxide reductase from the denitrifying bacterium Pseudomonas perfectomarina has been isolated and purified to homogeneity. The enzyme contained about eight copper atoms/120 KDa and was composed of two presumably identical subunits. The isoelectric point was 5.1.

Several spectroscopically distinct forms of the enzyme were identified. A ‘pink’ form of the enzyme was obtained when the purification was done aerobically. The specific activity of this species was around 30 nkat/mg protein as measured by the nitrous‐dependent oxidation of photochemically reduced benzyl viologen. A ‘purple’ form of the enzyme, whose catalytic acticity was 2–5‐fold higher, was obtained when the purification was done anaerobically. The activity of both forms of the enzyme was substantially increased by dialyzing the protein against 2‐(N‐cyclohexylamino)ethanesulfonate buffer at pH ∼ 10. A maximal activity of 1000 nkat/mg protein has been obtained for the purple form using this procedure. A ‘blue’, enzymatically inactive form of the enzyme resulted when either the pink or the purple species was exposed to excess dithionite or ascorbate.

Anaerobic, potentiometric titrations of both the purple and the pink form of the enzyme gave a Nernst factor, n540, of 0.95 and a midpoint potential, E′0.540 of +260 mV (vs SHE, 25°C, Tris/HCl buffer, pH 7.5).

Electron paramagnetic resonance (EPR) and optical spectra of N2O reductase suggested the presence of an unusual type 1 copper center. Type 2 copper was absent. The hyperfine splitting in the g‖ region consisted of a seven‐line pattern. In the presence of excess of reductant, a broad EPR signal with g values at 2.18 and 2.06 was observed. The EPR spectra of the pink and purple forms of the enzyme were similar; however, the spectrum of the purple form was better resolved with g‖= 2.18 (A‖= 3.83 mT) and g⊥= 2.03 (A⊥= 2.8 mT).

Most of the copper in N2O reductase was removed by anaerobic dialysis against KCN. Reaction of the apoprotein with Cu(en)2SO4 partially regenerated the optical and EPR spectra of the holoprotein; the resulting protein was enzymatically inactive.

Monospecific antibodies against the copper protein strongly inhibited the N2O reductase activity of purified samples and cell‐free extracts.
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.
Specific mRNP complexes
Authors: RUZDIJIC Sabera D. , BIRD R. Curtis , JACOBS Frederik A. , SELLS Bruce H.
Published: March 2005

These studies were designed to identify the proteins associated with specific mRNAs. L6 myoblasts contain a unique poly(A)‐rich H4 mRNA as well as poly(A)‐minus H4 mRNA subspecies. We have characterized the proteins present in both poly(A)‐rich and poly(A)‐minus histone H4 mRNP complexes following ultraviolet cross‐linking in vivo. In addition, the muscle‐specific myosin heavy chain (MHC) mRNP complex was characterized in myblasts. [35S] Methionine‐labelled poly(A)‐rich and poly(A)‐minus RNP complexes were prepared from both the polysomal and free (post‐polysomal) RNP compartments. From each fraction the mRNP encoding histone H4 or MHC was purified by hybrid selection to a cloned human histone H4 gene or MHC cDNA. A unique set of 6–16 proteins was found bound to each of the specific mRNP complexes. These proteins were a subset of the total population of either polysomal or free RNP proteins and some proteins appeared common among the different hybrid‐selected RNP fractions. The results demonstrate that (a) mRNAs bind a different set of proteins depending upon whether they are present in the polysomal or free mRNP fraction; (b) the presence of poly(A) sequences affects the proteins which bind to H4 mRNA in the free RNP compartment.
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.
Characterization of a glucoamylase G2 from Aspergillus niger
Authors: SVENSSON Birte , LARSEN Kjeld , GUNNARSSON Alf
Published: March 2005

Peptide fragments were generated by enzymic or chemical degradation of the small form, G2, and the large form, G1, of Aspergillus niger glucoamylase (EC 3.1.2.3). The G2 form was either identical to residues Ala1– Pro512 or to Ala1– Ala514 of the G1 polypeptide chain containing 616 amino acid residues. Structural analysis of the O‐linked carbohydrates from the 70‐amino‐acid‐residues long extensively glycosylated segment of G2 revealed no significant differences in the contents of single mannose and oligosaccharide units in comparison to the corresponding region of G1. The results suggest that the present G2 form has been generated by limited proteolysis of the larger G1. In contradistinction to this, a recently reported splicing out of an intervening sequence from G1 mRNA leads to a smaller mRNA coding for a G2 protein product with a different COOH‐terminal sequence than the G2 form described in the present work [Boel et al. (1984) EMBO J. 3, 1097–1102].
The complete amino acid sequence of vitelline coat lysin
Authors: HAINO‐FUKUSHIMA Kazu , KASAI Hisataka , ISOBE Toshiaki , KIMURA Masami , OKUYAMA Tsuneo
Published: March 2005

The vitelline coat lysin of a top shell, Tegula pfeifferi, is a single polypeptide consisting of 118 amino acid residues and having a relative molecular mass of 13800. The complete amino acid sequence of the vitelline coat lysin was determined by the analyses of five peptides obtained by cyanogen bromide degradation and three fragments obtained by Staphylococcus aureus protease digestion of the protein. The sequence showed the presence of microheterogeneities in the vicinity of the C‐terminal half of the molecule and the existence of two homologous domain structures.
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.
Expression of the polyoma middle‐size T antigen in Escherichia col
Authors: PALME Klaus , ECKHART Walter
Published: March 2005

We constructed a plasmid encoding a hybrid protein, consisting of the N‐terminal signal sequence of the major outer membrane lipoprotein (lpp) of Serratia marcescens joined to the polyoma middle‐size T antigen (mT antigen). The hybrid protein expressed under the control of a lpp‐lac hybrid promoter was synthesized at levels up to 5% of newly synthesized protein and could be accumulated in Escherichia coli strains carrying the Cap R mutation. The mT antigen produced in E. coli was precipitated by polyoma antitumor serum, and by serum directed against a synthetic peptide corresponding to the C terminus of the authentic mT antigen. The protein was secreted into the periplasmic space, from which it could be released by osmotic shock. The bacterial mT antigen had no detectable associated protein kinase activity.
Purification and characterization of ovine angiotensinogen
Authors: FERNLEY Ross T. , JOHN Marie , NIALL Hugh D. , COGHLAN John P.
Published: March 2005

The two major forms of ovine angiotensinogen (renin substrate) have been purified to homogeneity from plasma and a third form has been partially purified. The purification procedure involved ammonium sulphate fractionation, gel filtration, ion‐exchange chromatography and chromatofocusing. The pure proteins have apparent relative molecular masses of 56000 as determined by sodium dodecyl sulphate gel electrophoresis. The amino acid compositions of the two major forms appear to be the same; however, they do have different carbohydrate compositions. Antibodies raised against the a form showed complete cross‐reactivity with the b and c forms. Both major forms have the same amino‐terminal sequence, which includes that of ovine angiotensin I: Asp‐Arg‐Val‐Tyr‐Ile‐His‐Pro‐Phe‐His‐Leu. Thus ovine angiotensin is the Ile5 form and not the Val5 form as had previously been suggested.
Nuclear‐magnetic‐resonance studies on the conformation of membrane‐bound α‐mating factor
Authors: WAKAMATSU Kaori , OKADA Akihiko , SUZUKI Midoriko , HIGASHIJIMA Tsutomu , MASUI Yoshihiro , SAKAKIBARA Shumpei , MIYAZAWA Tatsuo
Published: March 2005

The C‐H proton resonances of α‐mating factor, yeast pheromone, in 2H2O solution were assigned. The phase transition temperature of perdeuterated dipalmitoylglycerophosphocholine (suspension) was found to be 35.5°C. In the presence of vesicles of this phospholipid, the exchange broadening and transferred nuclear Overhauser effect (TRNOE) of peptide proton resonances (at 50°C) were analyzed. The mode of binding of this peptide with the phospholipid bilayer was elucidated. The N‐terminal nine residues (Trp1– Gly9) are tightly bound to the bilayer, while the C‐terminal four residues (Gln10– Tyr13) are left free in aqueous phase. This is consistent with the previous observation that the C‐terminal three residues (Pro11– Tyr13) are not essential for the activity of this pheromone [Masui, Y. et al. (1977) Biochem. Biophys. Res. Commun. 78, 534–538]. Furthermore, from the TRNOE analyses, the conformation of the membrane‐bound N‐terminal part of α‐mating factor was elucidated: the residues Trp1– Gln5 form a compact helical structure while the residues Lys7– Gly9 form an extended structure. A similar TRNOE was also observed for an active decapeptide analog Trp1– Gln10. This confirms the previous conclusion that the physiological activities of this pheromone and analog peptides are correlated with the conformations of membrane‐bound peptide molecules [Higashijima, T. et al. (1983) FEBS Lett. 159, 229–232].
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.
Analysis of human p53 proteins and mRNA levels in normal and transformed cells
Authors: MATLASHEWSKI Greg , BANKS Lawrence , PIM David , CRAWFORD Lionel
Published: March 2005

p53 mRNA and proteins were examined in a variety of human transformed cells and in normal human foreskin fibroblast cells. Both the steady‐state and translatable levels of p53 mRNA were the same in normal and transformed human cells. In vitro synthesized p53, programmed by mRNA from normal and transformed human cells, revealed that there was heterogeneity in the primary structure of p53 from these cells. Pulse labeling of cells and immunoprecipitation analysis with a panel of human reactive anti‐p53 antibodies demonstrated that the types of p53 synthesized in vitro corresponded to the types made in vivo from SV80 and COLO 320 cells. No p53 was detectable by similar pulse‐labeling analysis of HeLa and normal foreskin fibroblast cells. Since it was necessary to use anti‐p53 sera from cancer patients to carry out much of the immunoprecipitation analysis in this study we therefore further characterised these sera to determine if they reacted with one or more than one epitope. p53‐β‐galactosidase fusion proteins were synthesized in Escherichia coli and used to analyse the anti‐p53 antibodies produced by cancer patients. We demonstrate that the antisera contain antibodies directed against epitopes in both the N‐terminal and C‐terminal regions of the p53 molecule.
Chain length heterogeneity of lipopolysaccharide released from Salmonella typhimurium by ethylenediaminetetraacetic acid or polycations
Authors: HUKARI Ritva , HELANDER Ilkka M. , VAARA Martti
Published: March 2005

Cells of two smooth Salmonella typhimurium strains (SL696 and SH4247) were treated with ethylenediaminetetraacetic acid (EDTA) and the polycations poly(L‐lysine) and protamine to monitor both quantitatively and qualitatively the release of [14C] galactose‐labelled lipopolysaccharide into the medium to find out whether these effector substances caused selective release of certain fractions from the initially heterogenous lipopolysaccharide population. Each one of the substances released considerable amounts of lipopolysaccharide into the medium. Analysis by sodium dodecyl sulphate/polyacrylamide gel electrophoresis followed by autoradiography showed that the total lipopolysaccharide (from isolated membranes) and the released materials produced coincident banding patterns, each with a high degree of O side‐chain length heterogeneity. Densitometric scans of the autoradiograms were analyzed for possible differences in the distribution and relative abundance of lipopolysaccharide molecules with different O chain lengths. It was found that in SL696 the released materials were identical to the total lipopolysaccharide; in SH4247 subtle deviations from the total lipopolysaccharide were seen. We conclude from these results that lipopolysaccharide molecules with short and long O side chains are linked to and stabilized in the outer membrane by similar mechanisms equally susceptible to the effects of EDTA and polycations.
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.
Enzymatic cyclization of all‐trans pentaprenyl and hexaprenyl methyl ethers by a cell‐free system from the protozoon Tetrahymena pyriformis
Authors: RENOUX Jean‐Marie , ROHMER Michel
Published: March 2005

A cell‐free system from the protozoon Tetrahymena pyriformis capable of cyclizing squalene into tetrahymanol cyclizes all‐trans pentaprenyl methyl ether to a scalarane‐type sesterterpene and all‐trans hexaprenyl methyl ether to bicyclo‐, tricyclo‐, tetracyclo‐ and pentacyclohexaprenyl methyl ethers, each corresponding to a possible cationic intermediate. The structures of the cyclization products have been determined by spectroscopic methods and are compatible with a biogenetic scheme involving polyprenyl ether cyclization. This is the first direct proof of an enzymatic cyclization of higher isoprenic alcohol derivatives, and we assume it was performed by the squalene‐to‐hopane cyclase of the protozoon. The formation of a scalarane‐type sesterterpene from C25 polyprenyl methyl ether suggests that these terpenoids, whose presence is restricted to a few sponges, might be in fact microbial metabolites. Tricyclopolyprenyl derivatives have been identified in the organic matter from numerous sediments and they were interpreted as being chemical fossils of still unidentified microorganisms. The cyclization of hexaprenyl methyl ether is the first attempt of identification of these tricyclopolyprenol derivatives in living organisms.
Production of anti‐(ADP‐ribose) antibodies with the aid of a dinucleotide‐pyrophosphatase‐resistant hapten and their application for the detection of mono(ADP‐ribosyl)ated polypeptides
Authors: MEYER Thomas , HILZ Helmuth
Published: March 2005

Previous attempts to produce anti‐(ADP‐ribose) antibodies by immunization of rabbits with ADP‐ribose conjugated to serum albumin had resulted in the production of 5′AMP‐specific antibodies [Bredehorst et al. (1987) Eur. J. Biochem. 82, 105–113]. To obtain true anti‐(ADP‐ribose) antibodies an antigen was constructed that was resistant to enzymic degradation at the pyrophosphate group. The enzymically active β‐methylene derivative of NAD (NAD[CH2]) was synthesized from ADP containing a methylene bridge (CH2) instead of an oxygen in the diphosphate group. NAD[CH2] was converted to its N6‐[(2‐carboxyethyl)thiomethyl] derivative and hydrolyzed to the corresponding ADP[CH2]‐ribose derivative which was then coupled to bovine serum albumin. The antibodies obtained with this antigen were specific for free or protein‐bound ADP‐ribose groups, except for a cross‐reaction with FAD. AMP, ADP, ATP or poly(ADP‐ribose) interfered with [3H]ADP‐ribose tracer binding only at higher concentrations. No interference was observed with poly(A), RNA and DNA at 6000‐fold excess. The antibodies were purified on a novel type of affinity matrix. This was formed from NAD and guanidinobutyrate by a cholera‐toxin‐catalyzed reaction and the product, ADP‐ribosyl guanidinobutyrate, was bound to Affi Gel by carbodiimide‐aided condensation. The purified antibodies allowed the detection of ADP‐ribose conjugated to polypeptides in amounts lower than 1 pmol as demonstrated by immunoblotting of [14C]ADP‐ribosylated elongation factor 2. They also could be used to observe in situ, by indirect immunofluorescence, the increased mono(ADP‐ribosyl)ation of nuclear proteins in dimethyl‐sulfate‐treated cells, and to show that histone H2B was the principal histone acceptor of single ADP‐ribose groups in alkylated 3T3 cells.
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.
Small cytoplasmic RNAs from human placental free mRNPs
Authors: LORBERBOUM Haya , DIGWEED Martin , ERDMANN Volker A. , SERVADIO Yael , WEINSTEIN Daniel , GROOT Nathan DE , HOCHBERG Abraham A.
Published: March 2005

A new family of small cytoplasmic RNA species (scRNAs) was found to be associated with human termplacental free messenger ribonucleoprotein particles (mRNPs). Placental scRNAs strongly inhibit translation of both homologous and heterologous mRNAs in a cell‐free rabbit reticulocyte system. scRNAs could be resolved into at least four different RNA species. One of the RNA molecules, scRNA species 1, was the most potent protein synthesis inhibitor found among the placental scRNAs. The nucleotide sequence of the scRNA species 1 was determined. In spite of its short length, scRNA species 1 still exhibited a very strong inhibitory effect on the in vitro protein synthesis. scRNAs were found to be complexed with proteins in the form of scRNPs. Proteins of these complexes enhanced the inhibitory effect of scRNAs on in vitro translation. Experiments provided evidence that inhibition of in vitro protein synthesis by the scRNAs is not dependent upon mRNA concentration. However, inhibition can be overcome by increasing the ratio lysate/scRNAs, thus suggesting that scRNAs act on some essential component of the cell‐free system. The degree of inhibition is decreased when scRNAs are added after the start of translation, suggesting that scRNAs (or scRNPs) interfere with the initiation stage of translation, probably acting on an initiation factor(s). Placental scRNAs are unique in their size, being smaller than other known scRNAs. Their association with free cytoplasmic repressed mRNPs in human placenta suggests that scRNAs play a role in the regulation of mRNP metabolism and, consequently, in the control of mRNA translation.
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.
Processing of human choriogonadotropin and its receptors by cultured pig Leydig cells
Authors: BERNIER Michel , CLERGET Magali , MOMBRIAL Charlotte F. , SAEZ José M.
Published: March 2005

We have examined the process by which human choriogonadotropin/luteinizing hormone (hCG/LH) receptors are regulated in cultured porcine Leydig cells. Treatment of Leydig cells with human choriogonadotropin, cholera toxin, forskolin and cyclic 8‐bromoAMP (8‐BrcAMP) produced a loss of surface receptors without modification of the binding affinity. This negative regulation of the number of receptors mediated by maximal concentrations of hCG was higher than that induced by the other agents. The extent of receptor loss in cells treated with increasing concentrations of hCG was highly correlated with their capacity to stimulate cAMP production. However, there was little correlation between down‐regulation and cAMP production of these cells treated by hCG plus forskolin or cholera toxin plus forskolin, where a synergistic cAMP production was obtained. Following exposure of Leydig cells to both hCG and 8‐BrcAMP, the surface receptor disappearance began after an initial lag period of about 6–8 h. Thereafter a 50% loss of surface receptor was observed in the next 8‐h incubation. Monensin with hCG shortens this lag period before initiation of receptor loss. Kinetic studies with 125I‐hCG, in the presence or absence of monensin, showed that the half‐life of the receptor‐bound hormone complexes at the cell surface was 10.5 h and 8 h respectively. Therefore, the steady state of the surface receptor during the lag phase of 8 h is probably related to recycling of internalized receptors and/or translocation of preformed receptors. Cycloheximide and actinomycin D inhibit hCG‐mediated and 8‐BrcAMP‐mediated down‐regulation. Cycloheximide lengthens ligand‐receptor complexes at the surface by slowing down the rate of internalization (half‐life of 20 h), but this mechanism is not enough per se to explain the effect of cycloheximide. Pulses of hCG or 8‐BrcAMP for 4 h and 8 h sufficed to induce nearly maximal down‐regulation. However, it was possible to attenuate this triggering effect by adding cycloheximide after pulse of the cells. Thus, even after removal of the triggering agent (hCG or 8‐BrcAMP), the loss of surface receptor could be triggered by a protein‐sensitive signal.

Taken as a whole these results indicate that a coordinated interaction is involved in the cell‐surface hCG/LH receptor regulation. The apparent steady state of the number of receptors during the first hours of stimulation passed through a reuptake of internalized receptors. It is also proposed that the stimulatory agent may deplete the surface pool of receptors by progressively preventing the return of internalized receptors to the surface by a process sensitive to the newly synthesized proteins, rather than by increasing their rate of internalization.
Rate constant for capping of the barbed ends of actin filaments by the gelsolin‐actin complex
Authors: SELVE Norma , WEGNER Albrecht
Published: March 2005

The rate of capping of actin filaments by the gelsolin‐actin complex was measured by inhibition of elongation of the barbed ends of actin filaments. Polymeric actin (0.1 – 1.0 μM) was added to 0.5 μM monomeric actin and various concentrations of the gelsolin‐actin complex (0.08–2.4 nM) to induce nucleated polymerization. As under the experimental conditions (2 mM MgCl2, 100 mM KCl, 37°C, actin monomer concentration ≤ 0.5 μM) actin filaments treadmilled, filaments elongated only at the barbed ends and the gelsolin‐actin complex did not nucleate actin filaments to polymerize towards the pointed ends. The rate of nucleated actin polymerization in the presence of the gelsolin‐actin complex was quantitatively analyzed. The rate constant for capping of the barbed ends of actin filaments by the gelsolin‐actin complex was found to be about 107 M−1 s−1.
Chemical modification of NADPH‐cytochrome P‐450 reductase
Authors: INANO Hiroshi , TAMAOKI Bun‐ichi
Published: March 2005

Chemical modification of rat hepatic NADPH‐cytochrome P‐450 reductase by sodium 2,4,6‐trinitrobenzenesulfonate (TNBS) resulted in a time‐dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo‐first‐order kinetics with a reaction order approximately one, and a second‐order constant of 4.8 min−1· M−1. The reducing activities for 2,6‐dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH‐cytochrome P‐450 reductase from inactivation by TNBS was acheived by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3‐Acetylpyridine‐adenine dinucleotide phosphate, adenosine 2′,5′‐bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2′‐phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH‐cytochrome P‐450 reductase.
Cytochalasin D alters the rate of synthesis of some HEp‐2 cytoskeletal proteins
Authors: TANNENBAUM Janet
Published: March 2005

1. The most abundant proteins of HEp‐2 cells were resolved by two‐dimensional gel electrophoresis. The protein spots corresponding to several cytoskeletal proteins (vimentin, α‐tubulin, β‐tubulin, α‐actinin, tropomyosins, and cytokeratins) were identified by comigration with protein markers or by immunological techniques.

2. After treatment of HEp‐2 cells with 0.2 μM or 2.0 μM cytochalasin D for 20 h, radioautograms of two‐dimensional gel patterns of lysates from cells pulse‐labeled with [35S]methionine indicated that the drug altered the rate of synthesis of some proteins. The relative rate of synthesis of the identified cytoskeletal proteins was measured. Synthesis of α‐actinin, the higher‐molecular‐mass pair of tropomyosins and actin were similarly increased with cytochalasin D treatment, suggesting coordinate induction. Vimentin and tubulin synthesis was depressed. One cytokeratin exhibited an increase in synthesis comparable to actin, another was increased to a lesser extent and one was decreased.