bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrogenic steps in the redox reactions catalyzed by photosynthetic reaction‐centre complex fromRhodopseudomonas viridis
Authors: DRACHEVA Stella M. , DRACHEV Lel A. , KONSTANTINOV Alexander A. , SEMENOV Alexey Yu. , SKULACHEV Vladimir P. , ARUTJUNJAN Alexander M. , SHUVALOV Vladimir A. , ZABEREZHNAYA Svetlana M.
Published: March 2005

Electrogenic and redox events in the reaction‐centre complexes fromRhodopseudomonas viridishave been studied. In contrast to the previous points of view it is shown that all the four hemes of the tightly bound cytochromechave different Emvalues (‐60, +20, +310 and +380 mV). The first three hemes reveal α absorption maxima at 554 nm, 552 nm and 556 nm respectively. The 380‐mV heme displays a split α band with a maximum at 559 nm and a shoulder at 552 nm. Such a splitting is due to non‐degenerated Qxand Qytransitions in the iron‐porphyrin ring as demonstrated by magnetic circular dichroism spectra. Fast kinetic measurements show that, at redox potentials when only high‐potential hemesc‐559 andc‐556 are reduced, hemec‐559 appears to be the electron donor to P‐960+(τ= 0.32 μs) whereas hemec‐556 serves to rereducec‐559 (τ= 2.5 μs). Upon reduction of the third heme (c‐552), the P‐960+reduction rate increases twofold (τ= 0.17 μs) and all photoinduced redox events within the cytochrome appear to be complete in less than 1 μs after the flash. The following sequence of the redox centers is tentatively suggested:c‐554,c‐556,c‐552,c‐559, P‐960.

To study electrogenesis, the reaction‐centre complexes fromRps. viridiswere incorporated into asolectin liposomes, and fast kinetics of laser flash‐induced electric potential difference has been measured in proteoliposomes adsorbed on a phospholipid‐impregnated film. The electrical difference induced by a single 15‐ns flash was found to be as high as 100 mV. The photoelectric response has been found to involve four electrogenic stages associated with (I) QAreduction by P‐960+(II) reduction of P‐960+by hemec‐559; (III) reduction ofc‐559 byc‐556 and (IV) protonation of Q2−B. The relative contributions of stages I, II, III and IV are found to be equal to 70%, 15%, 5% and 10%, respectively, of the overall electrogenic process. At the same time, the first three respective distances along the axis normal to the membrane plane covered by electrons, calculated from X‐ray data of Deisenhofer et al. [J. Mol. Biol. 180, 385–398 (1984)], are 22%, 18.5% and 26%. This indicates that the efficiency of electrogenic phases depends first of all upon the value of the dielectric constant of the respective membrane regions rather than upon the distance between the redox groups involved. The efficiency is higher, the deeper these groups are immersed in the membrane.
Rate of calcium release and ATP synthesis in sarcoplasmic reticulum vesicles
Authors: SANDE‐LEMOS M. Piedade , DE MEIS Leopoldo
Published: March 2005

Sarcoplasmic reticulum vesicles can catalyze the synthesis of ATP coupled to the efflux of calcium. The rate of this reaction is much faster when the vesicles are loaded in a medium containing phosphate than when oxalate is the precipitating agent. Two components of ATP synthesis can be observed when vesicles loaded with calcium phosphate are used. In the millisecond range and when the loaded vesicles are phosphorylated by Pi, the addition of ADP leads to an initial burst of ATP synthesis and after 50 ms approximately 3.0 nmol of ATP/mg protein are synthesized. This burst is not inhibited by ATP and is enhanced by physiological concentrations of KCl. The slow component of ATP synthesis is inhibited by both ATP and 100 mM KCl. In the physiological pH range, betaine, a trimethylamine present in different tissues, increases the level of phosphoenzyme formed by Pi and enhances the amount of ATP synthesized during the first turn of the reversal of the calcium pump.
The tubulin genes of Trypanosoma cruzi
Authors: MAINGON Rhaiza , GERKE Roger , RODRIGUEZ Margarita , URBINA Julio , HOENICKA Janet , NEGRI Stefania , AGUIRRE Tania , NEHLIN Jan , KNAPP Teresa , CRAMPTON Julian
Published: March 2005

The organization of the α‐ and β‐tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (α‐β)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating α‐ and β‐tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T.(Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruziβ‐tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesienseβ‐tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3‐kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.
Structural and functional studies of the interaction of the eukaryotic elongation factor EF‐2 with GTP and ribosomes
Authors: NILSSON Lars , NYGÅRD Odd
Published: March 2005

The structure of the guanosine nucleotide binding site of EF‐2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N‐chlorosuccinimide. Native EF‐2 contains two exposed trypsin‐sensitive cleavage sites. One site is at Arg66 with a second site at Lys571/Lys572. oGTP was covalently bound to the factor between Arg66 and Lys571. After further cleavage of this fragment with the tryptophan‐specific cleavage reagent N‐chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP · EF‐2 complex was capable of forming a high‐affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF‐2 indistinguishable from that produced by GTP. Although GTP could be substituted by non‐covalently linked oGTP in the factor and ribosome‐dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF‐2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF‐2 cleaved at Arg66 were unable to form the high‐affinity complex with ribosomes while retaining the ability to form the low‐affinity complex and to hydrolyse GTP. The second cleavage at Lys571/Lys572 was accompanied by a total loss of both the low‐affinity binding and the GTPase activity.
Association of glyceraldehyde‐3‐phosphate dehydrogenase with mono‐and polyribosomes of rabbit reticulocytes
Authors: RYAZANOV Alexey G. , ASHMARINA Lyudmila I. , MURONETZ Vladimir I.
Published: March 2005

It has been shown recently that glyceraldehyde‐3‐phosphate dehydrogenase (GAPD) is one of the three major RNA‐binding proteins of rabbit reticulocytes [Ryazanov, A. G. (1985) FEBS Lett. 192, 131–134]. It was suggested that, due to its RNA‐binding capacity, GAPD can form loose dynamic complexes with polyribosomes.

This communication reports that a considerable amount of GAPD activity can be found in the mono‐ and polyribosome fraction after sucrose gradient centrifugation of rabbit reticulocyte lysate. An increase of ionic strength, as well as the addition of exogenous RNA to the extract, result in the removal of GAPD from the complex with mono‐ and polyribosomes. It appears that GAPD forms the complex with polyribosomes due to the interaction with some exposed RNA regions of these structures. Although the interaction of GAPD with ribosomes is weak, it can be detected under physiological ionic conditions by the difference boundary sedimentation velocity technique.

Association of GAPD with mono‐ and polyribosomes can be prevented by a low concentration (10 μM) of NADH, but not NAD+. A nitrocellulose filter binding assay also shows that NADH has a stronger inhibitory effect on the enzyme‐RNA complex formation, as compared with NAD+.

We propose that the RNA‐mediated association of GAPD with mono‐and polyribosomes can provide compartmentation of the energy‐supplying system on these structures within the cell. This can maintain a high local concentration of ATP and GTP near the sites of protein synthesis.
Secondary structure and hydrogen bonding of crambin in solution A two‐dimensional NMR study
Authors: LAMERICHS Rudolf M. J. N. , BERLINER Lawrence J. , BOELENS Rolf , DE MARCO Antonio , LLINÀS Miguel , KAPTEIN Robert
Published: March 2005

The secondary structure of crambin in solution has been determined using two‐dimensional NMR and is found to be essentially identical to that of the crystal structure. The H–D exchange of most amide protons can be accounted for in terms of the hydrogen bonds found in the X‐ray structure. Exceptions are the amide protons of Cys‐4 and Ser‐6, which exchange more slowly than expected, and of Asn‐46 for which the exchange is faster. These results might be explained by a slightly different conformation of the C‐terminal region of the protein in solution. The slow exchange of the amides of Cys‐32 and Glu‐23 might be due to aggregation involving an extremely hydrophobic part of the protein in solution.
Higher oxidation states of prostaglandin H synthase
Authors: DIETZ Reiner , NASTAINCZYK Wolfgang , RUF Hans Heinrich
Published: March 2005

The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 °C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1= 1.4 × 107 M−1 s−1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+· FeIVO). Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2= 65 s−1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome‐c peroxidase, was assigned to a two‐electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+· which was formed by an intramolecular electron transfer from tyrosine to the porphyrin‐π‐cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.
Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules
Authors: DE MEIS Leopoldo , TUENA DE GÓMEZ PUYOU Marietta , PUYOU Armando GÓMEZ
Published: March 2005

The hydrophobic nature of the active site of two energy‐transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe · bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi⇆ ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi⇆ ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p‐trifluoromethoxy‐phenylhydrazone.

The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe · bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+‐ATPase.

The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic–hydrophilic transition during the catalytic cycle.
Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation
Authors: LEGRAND Chantal , DUBERNARD Véronique , KIEFFER Nelly , NURDEN Alan T.
Published: March 2005

The radiolabelled monoclonal antibody, 5G11, directed against native thrombospondin, has been used to assess the surface expression of secreted thrombospondin on human blood platelets. Emphasis has been placed on studying the role of fibrinogen in this process. Unstimulated platelets bound low amounts of 5G11 (about 2000 molecules/platelet). Binding increased 2‐fold and 5‐ 7‐fold after stimulation of platelets with ADP or thrombin (or ionophore A23187) respectively. Unstimulated platelets from patients deficient in α‐granule proteins (gray platelet syndrome) bound baseline levels of 5G11. However, binding was not increased after activation. Thrombospondin expression on thrombin‐stimulated normal platelets was for a large part divalent‐cation‐dependent and was not affected by AP‐2, a monoclonal antibody to GPIIb‐IIIa complexes. However, binding of 5G11 was some 50% lower when platelets were stimulated in the presence of Fab fragments of a polyclonal rabbit antibody to fibrinogen. This suggested either a direct binding of thrombospondin to surface‐bound fibrinogen or a steric inhibition due to a close proximity of the two proteins. The fact that binding of 5G11 was at the lower limit of the normal range to the stimulated platelets of an afibrinogenaemic patient specifically lacking detectable fibrinogen favoured the latter explanation. Thus, a major fibrinogen‐independent pathway for thrombospondin expression must exist.
Human heme oxygenase cDNA and induction of its mRNA by hemin
Authors: YOSHIDA Tadashi , BIRO Peter , COHEN Tirza , MÜLLER Rita M. , SHIBAHARA Shigeki
Published: March 2005

Hemin treatment increased both activity and mRNA level of heme oxygenase in human macrophages. Using poly(A)‐rich RNA prepared from human macrophages treated with hemin, we have constructed a cDNA library in the Okayama‐Berg vector. The human heme oxygenase cDNA was isolated by screening this library with a rat cDNA and was subjected to nucleotide sequence analysis. The deduced human heme oxygenase is composed of 288 amino acids with a molecular mass of 32800 Da. The homology in amino acid sequences between rat and human heme oxygenase is 80%. Like rat heme oxygenase, human enzyme has a putative membrane segment at its carboxyl terminus, which is probably essential for the insertion of heme oxygenase into endoplasmic reticulum. Both rat and human heme oxygenase have no cysteine residues. Recently we have shown that rat heme oxygenase is a heat‐shock protein [J. Biol. Chem. 262, 12889–12892 (1987)], and therefore we examined the effects of heat treatment on the induction of heme oxygenase in human macrophages and glioma cells. In contrast to hemin treatment, heat treatment had no apparent effects in either human cell line on the activity of heme oxygenase and its mRNA levels. These results suggest that human heme oxygenase may not be a heat‐shock protein.
Stress response of Tetrahymena pyriformis to arsenite and heat shock: Differences and similarities
Authors: AMARAL Margarida D. , GALEGO Lisete , RODRIGUES‐POUSADA Claudina
Published: March 2005

Molecular and cellular events associated with the response of Tetrahymena pyriformis to stress induced by sodium meta‐arsenite, have been examined by pulse‐labelling experiments. This stress agent induces the synthesis of two main groups of proteins, with molecular masses in the ranges 70–75 kDa and 25– 29 kDa, together with other proteins of molecular masses 92, 83, 46, 42 (two species), 36 and 35 kDa.

Comparison of the results with those of a previous study concerning the response of T. pyriformis to heat‐shock, shows that the two main groups of proteins, as well as the 92‐kDa and 35‐kDa species, which seem to be similarly induced by both types of stress, display similar or identical peptide maps. Other stress proteins seem to be either heat‐shock‐specific or arsenite‐specific.

Studies using actinomycin D suggest that the response to arsenite is controlled mainly at the transcriptional level, for the 70–75‐kDa group and 92‐kDa proteins, but it seems that the other arsenite‐induced proteins are subjected to transcriptional/translational control. In fact, results obtained by northern blotting, show that the mRNA coding for the 70‐kDa stress protein is present only in stressed cells, whereas the 27‐kDa‐coding mRNA is present both in stressed and in unstressed cells. Inhibition of translation by cycloheximide has shown that heat‐shock‐induced‐messengers are conserved under heat to be immediately translated upon removal of that inhibitor. Qualitatively similar results are obtained after prolonged treatments of T. pyriformis with arsenite and cycloheximide.

The most striking difference between the responses of T. pyriformis to these two stress conditions is that arsenite does not repress normal protein synthesis so drastically as heat shock. In addition, our results suggest that some arsenite‐induced messengers may be more stable than the corresponding heat‐shock‐induced messengers.
Purification and characterization of five forms of glutathione transferase from human uterus
Authors: Di ILIO Carmine , ACETO Antonio , DEL BOCCIO Gilberto , CASALONE Enrico , PENNELLI Alfonso , FEDERICI Giorgio
Published: March 2005

Five glutathione transferase (GST)forms were purified from human uterus by glutathione‐affinity chromatography followed by chromatofocusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide slab gel electrophoresis all forms resulted composed of two subunits of identical molecular size. GST V (pI 4.5)is a dimer of 23‐kDa subunits, GST I (pI 6.8)and GST IV (pI 4.9)are dimers of 24‐kDa subunits whereas GST II (pI 6.1)and GST III (pI 5.5)are dimers of 26.5‐kDa subunits. GST V accounts for about 85–90% of the activity whereas the other isoenzymes are present in trace quantities. On the basis of the molecular mass of the subunits, amino acid composition, substrate specificities, sensitivities to inhibitors, CD spectra and immunological studies, GST V appeared very similar to transferase π. Structural and immunological studies provide evidence that GST IV is closely releated to the less ‘basic’ transferase (GST pI 8.5)of human skin. Extensive similarities have been found between GST II and GST III. The comparison includes amino acid compositions, subunits molecular size and immunological properties. The two enzymes, however, are kinetically distinguishable. The data presented also indicate that GST II and GST III are related to transferase μ and to transferase Ψ of human liver. Even though GST I has a subunit molecular mass identical to GST IV, several lines of evidence, including catalytic and immunological properties, indicate that they are different from each other. GST I seems not to be related to any of known human transferases, suggesting that it may be specific for the uterus.
Synergistic stimulation of S‐adenosylmethionine decarboxylase activity by Ca2+ ionophore A23187, cholera toxin and 1‐oleoyl‐2‐acetylglycerol
Authors: OTANI Shuzo , MATSUI‐YUASA Isao , MIMURA‐SHIMAZU Yasuko , MORISAWA Seiji
Published: March 2005

The Ca2+ ionophore A23187 induced S‐adenosylmethionine decarboxylase in guinea‐pig lymphocytes, and cholera toxin stimulated the induction synergistically. The activator of protein kinase C, 1‐oleoyl‐2‐acetylglycerol, did not induce S‐adenosylmethionine decarboxylase activity but potentiated the enzyme activity induced by A23187 or by A23187 and cholera toxin. The addition of both A23187 and cholera toxin induced S‐adenosylmethionine decarboxylase, but the further addition of 1‐oleoyl‐2‐acetylglycerol or 12‐O‐tetradecanoylphorbol 13‐acetate did not potentiate the enzyme induction in protein kinase‐C‐down‐regulated cells that had been treated with 12‐O‐tetradecanoylphorbol 13‐acetate for 18 h. These results suggest that a Ca2+‐dependent pathway, other than that for protein kinase C, is essential for the induction of S‐adenosylmethionine decarboxylase and that a cAMP‐dependent pathway and also protein kinase C are involved in the potentiation of the induction.
Identification of cytoplasmic nodule‐associated forms of malate dehydrogenase involved in the symbiosis between Rhizobium leguminosarum and Pisum sativum
Authors: APPELS Michiel A. , HAAKER Huub
Published: March 2005

The malate dehydrogenase activity (EC 1.1.1.37), present in the cytoplasm of Pisum sativum root nodules, can be separated by ion‐exchange chromatography into four different fractions. Malate dehydrogenase activity present in the cytoplasm of roots elutes mainly as a single peak.

During nodule development an increase in malate dehydrogenase activity per gram of material was observed. This increase occurred concomitantly with the increase in nitrogenase activity.

The kinetic properties of the separated malate dehydrogenases of root nodule cytoplasm and root cytoplasm were studied. The Km values for malate (2.6 mM), NAD+ (27 μM), oxaloacetate (18 μM) and NADH (13 μM) of the dominant form of the root nodule cytoplasm are much lower than those of the dominant malate dehydrogenase root form (64 mM, 4.4 mM, 89 μM and 70 μM respectively). Binding of malate by the enzyme‐NADH complex from root nodules results in an abortive complex, thereby blocking the further reduction of oxaloacetate by NADH. The dominant root malate dehydrogenase does not form the abortive complex.

From the kinetic data it is concluded, first, that the root nodule forms of the enzyme are capable of catalysing at a high rate the reduction of oxaloacetate, to meet the demands for malate governed by the bacterioid and the infected plant cell. The second conclusion, drawn from the kinetic data, is that under physiological conditions the conversion of oxaloacetate can be controlled just by the malate concentration.

Consequently the major root nodule forms of malate dehydrogenase are able to allow a high flux of malate production from oxaloacetate but also to establish a sufficient oxaloacetate concentration necessary for the assimilation and transport of fixed nitrogen.
Purification and some properties of the corrinoid‐containing membrane protein from Methanobacterium thermoautotrophicum
Authors: SCHULZ Harald , ALBRACHT Simon P. J. , COREMANS Joanna M. C. C. , FUCHS Georg
Published: March 2005

The cytoplasmic membrane of the methanogenic archaebacterium Methanobacterium thermoautotrophicum does not contain cytochromes, but did contain a corrinoid protein of molecular mass about 33 kDa which, after treatment with 10 mg Triton X‐100/mg protein, was contained in a protein complex of about 500 kDa. Washed membranes from 1 g dry cells contained about 70 nmol of the cobamide factor III (5‐hydroxybenzimidazolyl cobamide) as the sole corrinoid. The corrinoid‐containing protein complex was purified and some of its properties were studied. According to several criteria it is an integral membrane protein complex. The corrinoid‐protein complex, after about 100‐fold purification, gave a single band on native PAGE and still had molecular mass of about 500 kDa. In SDS‐PAGE several subunits were observed: in addition to the corrinoid‐carrying subunit of about 33 kDa, other polypeptides of approximately 28 kDa, 26 kDa, and possibly 23 kDa were present. One mole of the purified 500‐kDa protein complex contained ≥ eight moles of the cobamide factor III. It was estimated that the corrinoid‐protein complex accounts for 8% of the membrane protein of M. thermoautotrophicum. The visible spectrum of the oxidized protein exhibited absorbance maxima at 547 nm, 511 nm, and a shoulder at 468 nm, which disappeared upon reduction with dithionite. The midpoint potential of this transition was around ‐145 mV (pH 7). With EPR a Co2+ signal was observed within ‐50 mV and ‐350 mV with a maximum around ‐200 mV. Possible reasons for the disappearance of the Co2+ signal at low redox potentials are discussed. The line shape of the Co2+ signal was similar to that of Co2+ in free corrinoids. The signal of Co2+ could also be evoked by reduction with 5 mM dithiothreitol. From the redox properties of the corrinoid membrane protein it may be expected that in vivo the cobalt may become reduced and reoxidized. Its possible function as an electron‐mediating membrane protein in the metabolism of methanogenic bacteria is discussed.
Structure function studies on the lipoate‐acetyltransferase–component‐X‐core assembly of the ox heart pyruvate dehydrogenase complex
Authors: HODGSON Jeffery A. , DE MARCUCCI Olga G. L. , LINDSAY J. Gordon
Published: March 2005

Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues.

Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex.

Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross‐linked by reaction with phenylene‐o‐bismaleimide in the presence of pyruvate and the subunits contributing to the products of cross‐linking were identified by immune blotting. Species with very high Mr containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations.

The types of cross‐linked E2 products formed suggested that two thiols, reactible with phenylene‐o‐bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.
The reconstitution of a hybrid histone octamer containing avian 110Cys‐des‐thio‐histone H3 and sea‐urchin 73Cys‐histone H4
Authors: GREYLING H. Johann , SEWELL B. Trevor , VON HOLT Claus
Published: March 2005

A hybrid histone octamer was reconstituted from eythrocyte H2A and H2B, avian [110Cys‐des‐thio]histone H3 and the sea‐urchin sperm [73Cys]H4 variant. [110Cys‐Des‐thio]histone H3 was prepared by reaction of natural H3 with Raney nickel. The ability of the hybrid octamer to crystallise to the same form as the natural octamer demonstrated that the chemical modification of cysteine to alanine in H3 and the mutation from threonine to cysteine in sperm H4 do not alter histone‐histone interactions in the octamer. Since the sulfhydryl groups of both H4 molecules are fully accessible to 5,5′‐dithiobis(2‐nitrobenzoate) these residues provide suitable sites for the introduction of a single cysteine‐specific label per H4 molecule in the octamer.
Expression of opioid genes in bovine seminal vesicles
Authors: PICCOLI Renata , PASANISI Alessandra , CARSANA Antonella , PALMIERI Marta , D'ALESSIO Giuseppe
Published: March 2005

In seminal vesicles, the organ producing most of seminal plasma in the bovine species, the pro‐opiomelanocortin and the proenkephalin genes are transcribed and translated, and their translation products processed into opioid peptides, which are secreted into the seminal plasma. By using a micro‐organ preparation of seminal vesicles we found that, after 20 h of incubation with labelled methionine, a multiplicity of opioids was produced. Among these, [Met]enkephalin and β‐endorphin were positively indentified, whereas in the newly formed secretion only [Met]enkephalin was detected. This may be correlated to the finding that the concentration of β‐endorphin in an extract of seminal plasma was one order of magnitude lower than that of [Leu]enkephalin and [Met]enkephalin. These findings expand the picture of the presence of opiod peptides in the male reproductive tract, indicating that they should have a role(s) in the physiology of reproduction, not only in the hypothalamus‐pituitary‐gonadal axis, determining the reproductive potential, but also in the so‐termed sex accessory glands, determining the actual events leading to reproduction. To our knowledge this is also the first case studied of opioid peptides produced as exocrine hormones.
A dimeric inhibitor or insect α‐amylase from barley
Authors: LÁZARO Ana , SANCHEZ‐MONGE Rosa , SALCEDO Gabriel , PAZ‐ARES Javier , CARBONERO Pilar , GARCÍA‐OLMEDO Francisco
Published: March 2005

A cDNA clone, designated pUP‐44, whose longest open reading frame codes for a protein that is homologous to the wheat α‐amylase inhibitors, has been isolated from a library obtained from developing barley endosperm. The deduced sequence for the mature protein, which is 122 residues long, is preceded by a sequence of 30 residues which has the typical features of a signal peptide. A closely corresponding protein, designated BDAI‐1, has been isolated from mature endosperm. BDAI‐1 behaves as a dimer and inhibits the α‐amylase from the insect Tenebrio molitor at concentrations that have no effect on salivary or pancreatic α‐amylases.
Molecular analysis of resveratrol synthase
Authors: SCHRÖDER Gudrun , BROWN John W. S. , SCHRÖDER Joachim
Published: March 2005

Resveratrol synthase (RS), a key enzyme in biosynthesis of stilbene‐type phytoalexins, catalyzes the formation of resveratrol from coumaroyl‐CoA and malonyl‐CoA. Two cDNA clones, pGSC1 and pGSC2, have been isolated from cDNA libraries established with poly(A)‐rich RNA from peanut (Arachis hypogaea) cell cultures specifically induced for RS. These cDNAs were used to identify two genomic clones (pGSG10 and pGSG11). Sequence analysis shows that the two clones overlap in a large stretch of nearly identical sequences, and that pGSG10 contains the 5′ and pGSG11 the 3′ end of RS genes. The sequences reveal a single intron, and the size of the predicted protein is 42.7 kDa, in close agreement with that observed in polyacrylamide gels (43 kDa). Chalcone synthase (CHS), a key enzyme of flavonoid biosynthesis, utilizes the same substrates as RS, but the product is different (naringenin chalcone). Comparison of RS with CHS consensus sequences shows that the two genes are related. Homology extends throughout the coding region, and the intron in RS is at the same position as a conserved intron in CHS. However, RS reveals a substantial number of amino acid differences to CHS in positions highly conserved in all CHS enzymes. It is proposed that the two proteins possess a common scaffold necessary for binding of the substrates and the type of enzyme reaction, and that the differences are responsible for the formation of different products.
Modifications of oxidative phosphorylations in mitochondria isolated from a mutant of Saccharomyces cerevisiae
Authors: MANON Stephen , GUERIN Martine
Published: March 2005

Mutants of Saccharomyces cerevisiae were isolated which supported two unlinked nuclear mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial one conferring paromomycin sensitivity. Mitochondria isolated from such a mutant exhibited modifications of several phosphate‐requiring functions: (a) kinetic parameters of the phosphate dependence of ATP synthesis were modified; (b) in the absence of phosphate the inner mitochondrial membrane exhibited a high proton leakage; (c) mutant mitochondria always exhibited a poor respiratory control and required tenfold more phosphate to reach a maximal state 3 of respiration; (d) phosphate transport, as measured by swelling experiments, was mersalyl‐insensitive and, consequently, state 3 of the respiration and ATP synthesis remained less mersalyl‐sensitive than in wild‐type mitochondria.

Analysis of the mitochondrial metabolism of diploid and segregant strains indicates that these modifications are related to the cryosensitive phenotype; however, at present, a cooperative effect of the mitochondrial mutation cannot be eliminated. It is proposed that the phosphate carrier itself or a regulatory element was modified.
Rabbit skeletal muscle microsomes contain two distinct phenylalkylamine‐binding sites
Authors: WERNET Wolfgang , SIEBER Manfred , LANDGRAF Wolfgang , HOFMANN Franz
Published: March 2005

Lu49888, a photoaffinity analog of verapamil, was used to identify specific binding sites for phenylalkylamines of calcium channels present in rabbit skeletal muscle microsomes. Direct binding equilibrium measurements and displacement curves of Lu49888 by its non‐radioactive analog yielded an apparent single class of binding sites with Kd and Bmax values of 16.5 nM and 7.5 pmol/mg respectively. Lu49888 was specifically incorporated into three proteins of apparently 165 kDa, 55 kDa and 33 kDa. Incorporation into the 55‐kDa protein was blocked by 10–50‐fold higher concentrations of unlabeled phenylalkylamines compared to incorporation into the 165‐kDa protein, suggesting that the 165‐kDa and 55‐kDa proteins contain a high and a low‐affinity verapamil‐binding site respectively. The photoaffinity‐labeled proteins were solubilized by 1% digitonin or 1% Chaps in roughly equal amounts. The 165‐kDa protein bound to wheat‐germ‐agglutinin(WGA)–Sepharose and sedimented in sucrose density gradients with the same constant as the purified dihydropyridine receptor, which has been reconstituted to a functional calcium channel. The 55‐kDa membrane protein did not bind to the WGA–Sepharose column and sedimented in sucrose density gradients with a lower s value than the 165‐kDa protein. The 165‐kDa but not the 55‐kDa membrane protein was specifically labeled by azidopine, the photoaffinity analogue of dihydropyridines. The 55‐kDa protein of the purified dihydropyridine receptor was not significantly labeled by Lu49888 showing that the 55‐kDa protein of the membrane is unrelated to the purified high‐affinity dihydropyridine receptor.
Gi affects the agonist‐binding properties of β‐adrenoceptors in the presence of Gs
Authors: MARBACH Irit , SHILOACH Joseph , LEVITZKI Alexander
Published: March 2005

Pertussis‐toxin‐catalyzed ADP‐ribosylation of Gi in S49 membranes, but not in S49AC− membranes, which lack Gs, induces a threefold reduction of isoproterenol affinity to the β‐adrenoceptors. A similar treatment of turkey erythrocyte membranes, which are devoid of functional Gi, has no effect on β‐agonist affinity to their β‐adrenoceptors. Non‐hydrolyzable analogs such as GTP[S] induce a larger decrease in β‐adrenoceptor affinity in S49 cells towards the agonist isoproterenol as compared to pertussis‐toxin‐catalyzed ADP‐ribosylation of Gi. These results suggest that Gi affects β‐adrenoceptor affinity to its agonist and that this interaction requires the presence of Gs. It seems, therefore, that Gi physically interacts with Gs to exert its effects on the receptor and probably on adenylate cyclase as well. Our ability to detect (a) the effect of pertussis‐toxin‐catalyzed ADP‐ribosylation in S49 cells on β‐agonist affinity and (b) the quantitative difference between the effect of pertussis toxin (approx. threefold) and GTP[S] (fivefold to sevenfold) depends on the use of a simple but rigorous method to study in detail the affinity of β‐agonists to their receptors. This method seems to be superior to the analysis of displacement curves as a means to examine receptor‐ligand interactions.
Regulation of angiotensin II receptors and steroidogenic responsiveness in cultured bovine fasciculata and glomerulosa adrenal cells
Authors: PENHOAT Armelle , JAILLARD Christine , CROZAT Anne , SAEZ José M.
Published: March 2005

The aim of the present study was to investigate the effect of several effectors on angiotensin II (A‐II) receptors and steroidogenic responsiveness in cultured bovine fasciculata cells. Treatment of adrenal cells for 24 h with A‐II (0.1 μM), corticotropin (1 nM), phorbol ester (PMA 0.1 μM), calcium ionophore A23187 (0.1 μM) and cyclic 8‐bromoAMP (1 mM) produced a loss of A‐II receptors whereas the A‐II antagonist [Sar1‐Ala8]A‐II (0.1 μM) led to a small but significant increase. The extent of the down‐regulation of receptors following maximal concentrations of A‐II was greater than that produced by the other agents. The effects of A‐II were dose‐dependent with a ID50 of 3 nM. Since cycloheximide and actinomycin blocked the down‐regulation of receptors, it seems likely that the effectors lead to the synthesis of certain proteins which inhibit the recycling of internalized receptors.

Pretreatment of adrenal cells with A‐II induced both homologous (90% decrease) and heterologous (corticotropin 83, PMA and ionophore 76% decrease) steroidogenic desensitization. However, the cAMP response to corticotropin of A‐II‐pretreated cells was higher (P < 0.001) than for control cells. Pretreatment with PMA and A23187 also resulted in both homologous and heterologous steroidogenic refractoriness but to a lesser degree than that induced by A‐II. In contrast, corticotropin‐pretreated cells responded normally to further stimulation with corticotropin or A‐II. Similarly pretreatment of bovine adrenal glomerulosa cells with A‐II (1 nM and 0.1 μM) and corticotropin (1 nM) also induced A‐II receptor loss and steroidogenic refractoriness.

The present findings indicate that, in contrast to the results reported in vivo in the rat, where A‐II leads to up‐regulation of its own receptors on glomerulosa cells and increases steroidogenic responsiveness, this peptide results in both down‐regulation and desensitization in cultured bovine fasciculata and glomerulosa cells. Our results also emphasize the absence of correlation between A‐II receptor loss and steroidogenic responsiveness.
Molecular pathology of scrapie‐associated fibril protein (PrP) in mouse brain affected by the ME7 strain of scrapie
Authors: HOPE James , MULTHAUP Gerd , REEKIE Laura J. D. , KIMBERLIN Richard H. , BEYREUTHER Konrad
Published: March 2005

Scrapie‐associated fibrils (SAF) are disease‐specific structures found in extracts of the brains of animals affected with scrapie. These structures are pathological aggregates of a normal host protein (PrP). Abnormal post‐translational modification of PrP has been suggested to explain its aberrant properties in scrapie‐affected brains and although there is a form of PrP in SAF indistinguishable in size from the protein in uninfected brain, lower‐molecular‐mass variants of PrP are also found in SAF fractions. We report the characterisation of the multiple forms of PrP found in SAF fractions purified from mouse brain affected by the ME7 strain of scrapie. The quantitively major forms of PrP in SAF prepared without the use of proteinase K have the amino‐terminal sequence Lys‐Lys‐Arg‐Pro‐Lys‐Pro‐Gly‐Gly‐, identical to that predicted for the amino‐terminus of normal mouse brain PrP. However N‐terminal cleavage of some PrP does occur in vivo within a domain of repetitive sequences at sites similar to but distinct from those cut by proteinase K in vitro. This suggests the conformation of the protein in aggregates in vivo does not differ extensively from that in detergent‐treated SAF in vitro. We conclude that the size diversity of PrP in SAF is only partly due to N‐terminal proteolysis and is independent of the proteolysis that occurs if proteinase K is used in the purification of SAF. Apart from proteolytic changes in the structure of PrP, we found a novel, as yet unidentified, amino‐acid derivative of the arginine residue at position 3 in mouse PrP, which may predispose PrP to form SAF.
Lipoamide dehydrogenase from Azotobacter vinelandii
Authors: WESTPHAL Adrie H. , de KOK Arie
Published: March 2005

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9–23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded from SDS gel electrophoresis of the cell‐free extract, stained for protein or used for Western blotting. After subcloning of the 14.7‐kb insert of this plasmid the structural gene could be located on a 3.2‐kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined.

The protein‐coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome‐binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three‐dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483–496], it appears that essential residues in all four domains have been conserved.

The enzyme is strongly expressed, although expression does not depend on the vector‐encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.
Characterization of human neutrophil leukotriene B4ω‐hydroxylase as a system involving a unique cytochrome P‐450 and NADPH–cytochrome P‐450 reductase
Authors: SUMIMOTO Hideki , TAKESHIGE Koichiro , MINAKAMI Shigeki
Published: March 2005

Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20‐hydroxyleukotriene B4 (20‐OH‐LTB4) by the 150000 × g pellet (microsomal fraction) of human neutrophil sonicate. The reaction required molecular oxygen and NADPH, and was significantly inhibited by carbon monoxide, suggesting that a cytochrome P‐450 is involved. The neutrophil microsomal fraction showed a carbon monoxide difference spectrum with a peak at 450 nm in the presence of NADPH or dithionite, indicating the presence of a cytochrome P‐450. The addition of LTB4 to the microsomal fraction gave a type‐I spectral change with a peak at around 390 nm and a trough at 422 nm, indicating a direct interaction of LTB4, with the cytochrome P‐450. The dissociation constant of LTB4, determined from the difference spectra, is 0.40 μM, in agreement with the kinetically determined apparent Km, value for LTB4 (0.30 μM). Such a spectral change was not observed with prostaglandins A1, E1 and F2α, or lauric acid, none of which inhibited the LTB4, ω‐hydroxylation. The inhibition of the LTB4, ω‐hydroxylation by carbon monoxide was effectively reversed by irradiation with monochromatic light of 450 nm wavelength. The photochemical action spectrum of the light reversal of the inhibition corresponded remarkably well with the carbon monoxide difference spectrum. These observations provide direct evidence that the oxygen‐activating component of the LTB4, ω‐hydroxylase system is a cytochrome P‐450. Ferricytochrome cinhibited the hydroxylation of LTB4 and the inhibition was fortified by cytochrome oxidase. An antibody raised against rat liver NADPH‐cytochrome‐P‐450 reductase inhibited both LTB4ω‐hydroxylase activity and the NADPH‐cytochrome‐c reductase activity of human neutrophil microsomal fraction. These observations indicate that NADPH–cytochrome‐P‐450 reductase acts as an electron carrier in LTB4ω‐hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH–cytochrome‐c reductase activity but not the LTB4ω‐hydroxylase activity of human neutrophil microsomal fraction, suggesting that cytochrome b5 does not participate in the LTB4‐hydroxylating system. These characteristics indicate that the isoenzyme of cytochrome P‐450 in human neutrophils, LTB4ω‐hydroxylase, is different from the ones reported to be involved in ω‐hydroxylation reactions of prostaglandins and fatty acids.
8‐(2‐Carboxymethylthio)‐cGMP, a site‐1‐selective compound for cGMP‐dependent protein kinase
Authors: LANDGRAF Wolfgang , RACK Michael , HEIL Wolfgang G. , HOFMANN Franz
Published: March 2005

The cGMP analogue 8‐(2‐carboxymethylthio)‐cGMP (CMT‐cGMP) was synthesized and its binding to cGMP‐dependent protein kinase (cGMP kinase) was studied. CMT‐cGMP bound at 4°C with an over 1400‐fold higher affinity to site 1 than to site 2 of the native enzyme with apparent Kd values of 4.1 nM and 5.9 μM, respectively. The apparent selectivity for site 1 was about threefold less with the autophosphorylated enzyme and about sixfold with the catalytically active fragment of cGMP kinase. The apparent selectivity was confirmed by determination of the dissociation of [3H]cGMP from cGMP kinase in the presence of 1 μM CMT‐cGMP at 4°C. The apparent site 1 selectivity was 250‐fold at 30°C under the conditions of the phosphotransferase assay. The apparent Kd values were 47 nM and 11.7 μM for site 1 and 2, respectively. CMT‐cGMP stimulated the phosphotransferase activity of native and autophosphorylated cGMP kinase with Km values of about 80 nM. About 60% of the total catalytic rate of cGMP kinase was obtained in the presence of 1 μM CMT‐cGMP and 0.13 mM Kemptide. The apparent Km values for ATP and Kemptide were not affected. However, CMT‐cGMP activated the enzyme to the same level as cGMP when 1.3 mM Kemptide was present. CMT‐cGMP and cGMP inhibited CAMP‐stimulated autophosphorylation of cGMP kinase with IC50 values of 0.7 μM and 2 μM, respectively. Neither compound stimulated autophosphorylation of cGMP kinase by itself.

These results indicate that CMT‐cGMP binds with high preference to site 1 of cGMP kinase and that occupation of site 1 may lead to expression of a partial enzyme activity.