bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for the repetitive domain structure of pig calpastatin as demonstrated by cloning of complementary DNA
Authors: Takano Emiko , Maki Masatoshi , Hatanaka Masakazu , Mori Hirotaka , Zenita Koichi , Sakihama Toshiko , Kannagi Reiji , Marti Thomas , Titani Koiti , Murachi Takashi
Published: July 2002

A clone of complementary DNA (cDNA) coding for pig heart calpastatin was isolated using synthetic oligonucleotide probes. The amino acid sequence deduced from the nucleotide sequence revealed the occurrence of a repetitive sequence at the interval of 140 amino acids, substantiating the multidomain structure of calpastatin. A portion of the sequence of 251 amino acid residues predicted for pig heart calpastatin (107 kDa) was found to be identical with that of a peptide fragment derived from pig erythrocyte calpastatin (68 kDa) and sequenced by Edman degradation.
ATP can promote activation and deactivation of the rod cGMP‐phosphodiesterase
Authors: Kamps K.M.Paul , Hofmann Klaus Peter
Published: July 2002

The AT (amplified transient) signal is a flash‐induced increase of the near‐infrared light scattering from isolated bovine rod outer segments and is interpreted as a monitor of cGMP‐phosphodiesterase activation [(1985) FEBS Lett. 188 15‐20]. We have investigated the effects of ATP and cyclic GMP on this signal. It has been found that ATP enhances the AT signal the relative effect being the largest for low photoexcitation (~ 1 rhodopsin per disc membrane). At a high rhodopsin turnover which saturates the AT amplitude the effect of ATP is to accelerate the rise of the signal. ATP can also accelerate the falling phase of the signal. This deactivating effect depends on the simultaneous presence of cyclic GMP. The results indicate that ATP acts on the phosphodiesterase activation cycle promoting activation as well as deactivation dependent on cGMP as a cofactor.
Regulation of protein synthesis in rabbit reticulocyte lysates
Authors: Roberts Richard C. , Ranu Rajinder S.
Published: July 2002

The specificity of the heme‐regulated protein kinase (HRI) was investigated further by utilizing the isolated 38000 Da subunit (α subunit) polypeptide of eIF‐2 as the substrate. For this purpose, the three subunit polypeptides of eIF‐2 (38000 Da, α, 50000 Da, β; and 52000 Da, γ) were resolved by reversed‐phase high performance liquid chromatography (HPLC). Results show that HRI is incapable of phosphorylating the 38000 Da subunit separated from the other two eIF‐2 polypeptides. Data suggest that the substrate'specificity of HRI is determined by the quaternary structure assumed by the α subunit in association with the other two subunits in the eIF‐2 holoprotein.
Structure of a Cephalosporium acremonium mtDNA replicator
Authors: Peñalva Miguel A. , Patiño Cristina , Rubio Víctor
Published: July 2002

We have investigated the ARS (autonomously replicating sequence) activity of a 1.94 kb mitochondrial DNA fragment of Cephalosporium acremonium and found that several subfragments of this piece of mtDNA conferred the ARS phenotype. The nucleotide sequence of the fragment shows: (i) a high A + T content (72.5%); (ii) a perfect consensus ARS sequence (ATTTATATTTA) in the subfragment with the highest ARS activity; (iii) a large number of ARS consensus‐related sequences in the other subfragments, even in one lacking ARS activity; (iv) several potential hairpin structures. One of them contains the perfect consensus ARS sequence.
Purification and characterization of low potential c cytochromes from Desulfovibrio desulfuricans membranes
Authors: Kramer Jeffrey F. , Pope Daniel H. , Salerno J.C.
Published: July 2002

Desulfovibrio desulfuricans grown in a lactate‐sulfate medium produces, in addition to soluble cytochromes, c‐type cytochromes which appear to be integral membrane proteins. Two cytochromes can be separated, an abundant 15 kDa cytochrome and a 22 kDa cytochrome. Both have optical spectra characteristics of c‐type cytochromes. The 15 kDa cytochrome shows two n = 1 components in potentiometric redox titrations with midpoint potentials at −130 and −270 mV in the membrane; both were slightly lower in detergent‐solubilized preparations. We suggest a designation of cytochrome cc
m for this species. Its properties suggest a function as a transmembrane electron carrier between hydrogen and sulfate.
Structure of arthropod hemocyanin
Authors: Bak Henk J. , Neuteboom Ben , Jekel Peter A. , Soeter Nell M. , Vereijken Johan M. , Beintema Jaap J.
Published: July 2002

Hemocyanins are large multi‐subunit copper proteins that transport oxygen in many arthropods and molluscs. The amino acid sequence of subunit a of Panulirus interruptus hemocyanin (657 residues) has been completed and fitted to the electron‐density map (3.2 Å resolution). Comparison of amino acid sequence data for several different hemocyanin subunits of arthropod species indicated that the general features of the polypeptide architecture as found in spiny lobster hemocyanin occur in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, 540–600 million years ago.
Differentiation of creatine kinase MB and IgA‐linked BB isoenzymes on electrophoresis
Authors: Chen Ann , Wong Shan S.
Published: July 2002

IgA‐linked creatine kinase (CK, EC 2.7.3.2) is a macro CK type 1 isoenzyme that has an identical electrophoretic mobility to CK‐MB. Its presence has the potential of causing misdiagnosis of myocardial infarction. Mixing anti‐CK‐B antiserum with the sample prior to electrophoresis did not unequivocally distinguish between the two isoenzymes. Similarly, anti‐human IgG and IgM antibodies were also ineffective. However, the IgA‐linked isoenzyme band was removed by anti‐human IgA antiserum. While anti‐CK‐M antibodies did not affect the electrophoretic mobility of IgA‐linked CK‐BB, the antibody eliminated both the CK‐MB and CK‐MM bands. Thus, specific anti‐IgA and anti‐CK‐M antibodies may be used to establish the presence of the myocardial isoenzyme.
Proton translocation coupled to methanogenesis from methanol + hydrogen in Methanosarcina barkeri
Authors: Blaut Michael , Müller Volker , Gottschalk Gerhard
Published: July 2002

Addition of methanol to resting cells of Methanosarcina barkeri incubated under an atmosphere of molecular hydrogen resulted in an acidification of the medium. This acidification was not observed when H2 was replaced by N2 or air, or when the uncoupler tetrachlorosalicylanilide was present. 2‐Bromoethanesulfonate completely inhibited both methanogenesis and proton extrusion. N,N′‐Dicyclohexylcarbodiimide, an inhibitor of the proton‐translocating ATPase in M. barkeri, did not affect proton extrusion. Therefore, it could be concluded that proton translocation was coupled to the terminal methylcoenzyme M methylreductase reaction and that it was not due to an H+‐translocating ATPase. A maximum value of 4 H+ translocated per CH4 formed was calculated.
Amino acid sequence of a 32‐residue region around the thiol ester site in duck ovostatin
Authors: Nagase Hideaki , Brew Keith
Published: July 2002

To obtain the amino acid sequence at the thiol ester site in duck ovostatin for comparisons with other proteins, the native ovostatin was labeled with 14CH3NH2 at the reactive thiol ester site. The modified protein was reduced, carboxymethylated, and digested with trypsin. 14C‐labeled peptides isolated by gel filtration with Sephadex G‐50, ion‐exchange chromatography on DEAE‐cellulose and HPLC were subjected to automated sequence analysis, and the stretch of 32 amino acid residues containing the 14CH3NH2‐binding site were determined. A comparison of this sequence with the corresponding sequences in α2‐macroglobulin, and complement components C3 and C4 revealed 72, 31 and 34% homology, respectively. The results indicate that ovostatin is a close relative to plasma α‐macroglobulins and may share a common ancestor with C3 and C4.
Fragmentation of chloroplast coupling factor in dependence of bound nucleotides Preparation of a reconstitutionally active form of subunit δ
Authors: Engelbrecht S. , Junge W.
Published: July 2002

Previous studies on the ability of CF1, fragments to reconstitute photophosphorylation in CF1,‐depleted thylakoids have shown that the degree of reconstitution was correlated with the presence of subunit δ in the fragment. This was taken as evidence that subunit δ was necessary for plugging the active proton channel CF0 [(1986) Eur. J. Biochem. 160, 635–643]. We questioned whether or not δ alone had this ability. In order to obtain δ we investigated the role of bound nucleotides in the stability of CF1. Starting from ammonium sulfate‐precipitated CF1, we found that a low content of bound ADP (1 mol ADP/mol CF1) seemed to stabilize the β—δ interaction, while loosening the interaction between α,β and γ. By elution from an anion‐exchange column in the presence of the nonionic surfactant Mega 9 we obtained β3δ and CF1(—δ) (both containing one ADP) or, after washing with alcohol/glycerol mixtures, β (nucleotide‐free) and CF1/CF1(—ϵ). On the other hand, with a further 2 ADP and 2 ATP bound to CF1, (after incubation with excess ATP) the α‐β‐γ interaction was stabilized in such a way that subunit δ alone could be isolated from the complex. Subunit δ, when isolated by this procedure and added back to CF1‐depleted thylakoids, reconstituted a high rate of photophosphorylation.
Last step in the cooperative pathway for galactolipid synthesis in spinach leaves: Formation of monogalactosyldiacylglycerol with C18 polyunsaturated acyl groups at both carbon atoms of the glycerol
Authors: Oursel A. , Escoffier A. , Kader J.C. , Dubacq J.P. , Trémolières A.
Published: July 2002

In this paper, the formation of monogalactosyldiacylglycerol (MGDG) with C18 polyunsaturated acyl groups at both positions of the glycerol from phosphatidylcholine (PC) of liposomes was demonstrated by a three‐step procedure: spinach chloroplasts were first incubated with liposomes of PC‐containing polyunsaturated fatty acids at both positions of the glycerol in the presence of phospholipid transfer protein. Chloroplasts were then incubated with phospholipase C in order to form 1,2‐diacylglycerol (1,2‐DAG) from PC integrated in the chloroplast envelope. Finally, washed chloroplasts were incubated with UDP‐gal and MGDG was formed by galactosylation of 1,2‐DAG.
The long 3′‐untranslated regions of the PDGF‐A and ‐B mRNAs are only distantly related
Authors: Hoppe J. , Schumacher L. , Eichner W. , Weich H.A.
Published: July 2002

A cDNA clone of about 2300 base pairs was prepared from the human osteosarcoma cell line U‐2 OS by hybridization with a 22‐mer oligonucleotide complementary to the NH2‐terminus of PDGF‐A. Restriction and sequence analysis showed that this clone contains the entire coding region for PDGF‐A and a long 3′‐untranslated region which is only distantly related to that in the mRNA of PDGF‐B.
Phosphorylation of brush border myosin by brush border calmodulin‐dependent myosin heavy and light chain kinases
Authors: Rieker James P. , Collins Jimmy H.
Published: July 2002

Calmodulin‐dependent myosin light chain kinase isolated from chicken intestinal brush border phosphorylates brush border myosin at an apparently single serine identical to that phosphorylated by smooth muscle myosin light chain kinase. Phosphorylation to 1.8 mol phosphate/mol myosin activated the myosin actin‐activated ATPase about 10‐fold, to about 50 nmol/min per mg. Myosin phosphorylated on its light chains could then be further phosphorylated to a total of 3.2 mol phosphate per mol by brush border calmodulin‐dependent heavy chain kinase. Heavy chain phosphorylation did not alter the actin‐activated ATPase of either myosin prephosphorylated on its light chains or of unphosphorylated myosin.
Fate of polyoma origin of replication after its direct introduction into mice
Authors: Cohen Hannah , Benvenisty Nissim , Reshef Lea
Published: July 2002

Recently we have developed a method for direct introduction of calcium phosphate‐precipitated DNA into newborn rats. To examine whether the foreign DNA can replicate, a plasmid containing a polyoma origin of replication was injected into newborn mice. The plasmid was found intact in liver and spleen and able to transform bacteria. The foreign DNA had disappeared by the seventh day after injection. Yet, the plasmid DNA containing the polyoma origin of replication had undergone replication in both the liver and the spleen.
A comparative study of the reduction of EPR signal IIslow by iodide and the iodo‐labeling of the D2‐protein in photosystem II
Authors: Takahashi Yuichiro , Styring Stenbjörn
Published: July 2002

The reduction kinetics of EPR signal IIslow in the presence of iodide were measured with EPR spectroscopy and the amount of 125I incorporated into the D2‐protein in the reaction center of photosystem II was quantitated in a PS II core complex preparation. The halftime of signal IIslow reduction in the dark was estimated at 31, 25 and 20 min in the presence of 0, 20 and 50 mM KI, respectively, reflecting the reduction of signal IIslow by iodide. The amount of 125I that was incorporated into the D2‐protein in the dark was correlated to the reduction of signal IIslow by iodide. This correlation indicates that the site of iodide oxidation in the dark and the component giving rise to EPR signal IIslow are located on the D2‐protein in the PS II reaction center.
Histone acetyltransferase activity during the cell cycle
Authors: Golderer Georg , Loidl Peter , Gröbner Peter
Published: July 2002

Histone acetyltransferase activity was measured in isolated nuclei during the synchronous cell cycle of the myxomycete Physarum polycephalum. Nuclei were incubated with [14C]acetyl‐coenzyme A and an excess of exogenous calf thymus histones. The activity is periodic during the cell cycle; it rises during the S‐phase to reach a maximum in the early G2‐period with a decline in mid and late G2. Comparison of the pattern of enzyme activity with the in vivo acetylation of histones during the cell cycle reveals that the enzyme activity does not wholly determine the acetylation state, indicating that other factors, including possibly the structural state of chromatin, are responsible for the observed cell cycle pattern of in vivo histone acetylation.
Inhibition of insect juvenile hormone synthesis by phorbol 12‐myristate 13‐acetate
Authors: Feyereisen René , Farnsworth Dan E.
Published: July 2002

The synthesis of insect juvenile hormone III (JH III) by isolated corpora allata of the cockroach Diploptera punctata incubated in vitro is inhibited by phorbol 12‐myristate 13‐acetate (PMA), phorbol 12,13‐dibutyrate and 1‐oleyl‐2‐acetylglycerol. 4α‐Phorbol 12,13‐didecanoate and diolein are inactive. The inhibitory effect of phorbol 12‐myristate 13‐acetate is fully reversed by 2E,6E‐farnesol or by 2E,6E‐farnesoic acid. It is highest in corpora allata that are past their peak in secretory activity or that have been inhibited by injections of 20‐hydroxyecdysone. This effect of phorbol esters implicates protein kinase C in the regulation of insect corpus allatum activity.
Haloperidol‐succinylglycyl[125I]iodotyrosine, a novel iodinated ligand for dopamine D2 receptors
Authors: Garrigues A.M. , Gehelmann F. , Girault J.M. , Delaage M. , Labouesse J.
Published: July 2002

A novel radioiodinated ligand of the butyrophenone type has been synthesized for the quantification and characterization of dopamine D2 receptors. This haloperidol‐derived ligand, haloperidol‐succinylglycyl‐[125I]iodotyrosine ([125I]HSGTI), binds rapidly (equilibrium is reached within 30 min, at 10 pM and 37°C) and with high affinity (K
d=0.3 nM) to bovine striatal membranes. Its pharmacology, determined by competitive displacement with dopaminergic and non‐dopaminergic drugs, is characteristic of binding to dopamine D2 receptors.
Activity of proteoliposomes containing cytochrome oxidase in the submitochondrial orientation
Authors: Cooper C.E. , Nicholls P.
Published: July 2002

Cytochrome‐c oxidase proteoliposomes containing internally trapped cytochrome c can turn over on internal or external cytochrome c. At low TMPD levels the internal activity is significantly lower than the external activity as the functional internal cytochrome c is not fully reduced in the steady state. Increasing TMPD concentration increases the internal rate to equal that of the external enzyme. Internal activity results in the accumulation of TMPD+. Valinomycin increases this accumulation and subsequently FCCP decreases it. In the presence of excess external cytochrome c, the effects of these ionophores are reversed. The internally‐facing enzyme is thus capable of generating a ΔμH+ in proteoliposomes as well as in submitochondrial particles.
Primary structure of elongation factor 1γ from Artemia
Authors: Maessen G.D.F. , Amons R. , Zeelen J.P. , Möller W.
Published: July 2002

Complementary DNA corresponding to elongation factor 1γ, which forms a complex with EF‐1β, has been cloned. A λgt11 cDNA library has been screened with an antiserum against EF‐1βγ. The derived amino acid sequence of EF‐1γ corresponds to 429 amino acids excluding the initiator methionine, which is absent in the mature protein. About half of the protein was sequenced by direct protein sequence analysis. No clear homology with any other protein was found.
Molecular evolution of the calmodulin gene
Authors: Nojima Hiroshi
Published: July 2002

On the basis of the intron/exon organization and the intramolecular homology of DNA sequences, I propose a novel model for genesis of the calmodulin gene. A primordial calmodulin gene consisting of 51 base pairs (17 amino acids) was subjected to three‐fold duplication to create modern calmodulin with four calcium‐binding subdomains. The model elucidates the seemingly enigmatic positions of splice junctions observed in calmodulin genes.
Differential expression of an α‐tubulin gene during the development of Physarum polycephalum
Authors: Monteiro Mervyn J. , Cox Robert A.
Published: July 2002

The expression of an α‐tubulin gene (altB1 (NαTu) [(1987) J. Mol. Biol. 193, 427‐438]) of Physarum polycephalum (strain CLdAXE) was found to be governed by a developmental switch since mRNA transcripts were detected, by S1 nuclease analysis, in the plasmodial but not the amoebal phase of the life‐cycle. The conclusion that the altB1 (NαTu) allele codes for a plasmodial specific α‐tubulin isotype is supported by recent amino acid sequence data.
Phosphatidylinositol degradation in ischemic brain specifically activated by synaptosomal enzymes
Authors: Wikieł Hanna , Strosznajder Joanna
Published: July 2002

The effect of ischemia on phosphatidylinositol (PI) degrading enzymes in brain homogenate, cytosol and synaptosomes was investigated to evaluate the involvement of cholinergic receptor in PI hydrolysis during ischemia. The activity of phospholipase C in cytosol was not changed by ischemic insult. PI degradation in synaptosomes was significantly (60%) enhanced by ischemia. Atropine and γ‐butyrolactone abolished the ischemic effect on PI degradation, suggesting that the cholinergic receptor system mediates breakdown of PI during ischemia.
Purification of valyl‐tRNA synthetase high‐molecular‐mass complex from rabbit liver
Authors: Motorin Yu.A. , Wolfson A.D. , Orlovsky A.F. , Gladilin K.L.
Published: July 2002

A high‐molecular‐mass complex containing valyl‐tRNA synthetase has been purified to homogeneity from rabbit liver. The molecular mass of the complex is about 800 kDa. The complex consists of four polypeptides of 130, 50, 40 and 30 kDa.
The complete amino acid sequence of the ribosomal ‘A’ protein (L12) from Bacillus stearothermophilus
Authors: Garland W.G. , Louie K.A. , Matheson A.T. , Liljas A.
Published: July 2002

The complete amino acid sequence of the ribosomal ‘A’ protein (Bst L12) has been determined from Bacillus
stearothermophilus. The protein contains 122 amino acids and has a composition of Asp4, Ans3, Thr6, Glu20, Gln2, Pro3, Gly9, Ala23, Val13, Met2, Ile11, Leu8, Phe2, Lys15, Arg1 and a molecular mass of 12737 Da.
Phenylhydrazine as probe for cofactor identification in amine oxidoreductases Evidence for PQQ as the cofactor in methylamine dehydrogenase
Authors: van der Meer Robert A. , Jongejan Jacob A. , Duine Johannis A.
Published: July 2002

Homogeneous methylamine dehydrogenase (primary‐amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.3, MADH) from the bacterium Thiobacillus versutus was treated with the inhibitor phenylhydrazine (PH). Derivatization of the cofactor in MADH took place in a fast reaction to give compound I. A different product, compound II, was formed in a slow reaction at high O2 concentrations. The compounds I and II could be removed from the protein by proteolysis with pronase and purified to homogeneity. Products showing identical absorption spectra and chromatographic behaviour were isolated from the reaction mixture after incubatng pyrroloquinoline quinone (PQQ) with PH. Upon dissolving in dimethyl sulphoxide, both the enzyme‐derived as well as the model‐system‐derived compounds I and II were nearly quantitatively transformed into PQQ. The conclusion is, therefore, that MADH from T. versutus contains covalently bound PQQ, removable from the protein with pronase, and not a structural analogue of this cofactor without the carboxylic acid groups, as was recently proposed for MADH from Bacterium W3A1 [(1986) Biochem. Biophys. Res. Commun. 141, 562–568]. The properties of compounds I and II suggest that they are the ‘azo adduct’ and the ‘hydrazone adduct’ of PH and PQQ at the C(5)‐position, respectively. The finding that the reaction of a hydrazine with PQQ can lead to two different products, in enzymes as well as in a model system, has important implications for the interpretation of recent comparative studies aimed at detection of PQQ in amine oxidoreductases with Raman spectroscopy.
In vitro evidence that the coat protein is the programming factor in alfalfa mosaic virus‐induced RNA synthesis
Authors: Houwing Corrie J. , Jaspars E.M.J.
Published: July 2002

Viral RNA‐dependent RNA polymerase preparations were isolated from cowpea mesophyll protoplasts 26 h after inoculation with the complete genome (RNAs 1, 2 and 3) and the incomplete genome (RNAs 1 and 2) of alfalfa mosaic virus. Although in incompletely inoculated protoplasts the ratio minus‐/plus‐strand RNA synthesis is much higher than in completely inoculated ones [(1983) Virology 124, 75–85)], both enzyme preparations produced largely plus‐strand RNA on their endogenous templates. When made template‐dependent both were strongly inhibited by small amounts of coat protein. The experiments show that among the two proteins encoded by RNA 3 only the (parental) coat protein can be responsible for the programming of the viral RNA polymerase to plus‐strand synthesis.
Interaction of seminalplasmin with chlortetracycline, a fluorescent chelate probe of Ca2+
Authors: Shivaji Sisinthy
Published: July 2002

The interaction of seminalplasmin with chlortetracycline, a fluorescent chelate probe of Ca2+, was studied. The results indicate that seminalplasmin binds to chlortetracycline. The binding is not influenced by salt. Both Ca2+ and seminalplasmin probably bind to the same site on chlortetracycline. Seminalplasmin also reduced the Tb3+‐associated fluorescence of bovine spermatozoal plasma membrane. These results are discussed in relation to the inhibitory effect of seminalplasmin on the uptake of Ca2+ in bovine spermatozoa.
Phosphorylation of a gelatin‐binding protein from L6 myoblasts by protein kinase C
Authors: Cates G.A. , Litchfield D.W. , Narindrasorasak S. , Nandan D. , Ball E.H. , Sanwal B.D.
Published: July 2002

A gelatin‐binding glycoprotein from L6 rat myoblasts, designated gp46, was shown to be phosphorylated in vivo. This phosphorylation was increased slightly (18%) by phorbol ester treatment of L6 suggesting protein kinase C involvement. Purified gp46 could be phosphorylated in vitro with protein kinase C, but not by the catalytic subunit of cAMP‐dependent protein kinase. Comparison of the phosphotryptic peptide maps of in vitro and in vivo labeled gp46 suggested that in vivo phosphorylation of gp46 may be mediated by protein kinase C.
Lipoxin A augments release of thromboxane from human polymorphonuclear leukocyte suspensions
Authors: Conti Pio , Reale Marcella , Cancelli Alessandro , Angeletti Pietro U.
Published: July 2002

Lipoxin A (LXA) is a novel eicosanoid, generated by the interactions of lipoxygenases, which has a variety of biological actions. When added to human polymorphonuclear leukocytes, LXA stimulated thromboxane formation which was monitored as TxB2 by radioimmunoassay. The compound augmented the formation of TxA2 stimulated by the ionophore of divalent cations (A23187). Formation of thromboxane was inhibited by two non‐steroidal anti‐inflammatory drugs (i.e. indomethacin and proglumetacin). Results of the present study indicate that LXA can provoke the release and transformation of endogenous arachidonic acid to thromboxane. Moreover, they suggest a relationship between lipoxin A and the formation of cyclooxygenase pathway products.
Phosphoglycerate kinase from the extreme thermophile Thermus thermophilus Crystallization and preliminary X‐ray data
Authors: Littlechild J.A. , Davies G.J. , Gamblin S.J. , Watson H.C.
Published: July 2002

The glycolytic enzyme phosphoglycerate kinase has been prepared from the extreme thermophile Thermus thermophilus. In contrast to its eukaryote equivalents (yeast and horse muscle) this prokaryotic enzyme crystallizes from ammonium sulphate in the presence of nucleotide substrates. These crystals are of a form and size well suited to high resolution X‐ray diffraction study using rotation camera techniques.
Degradation of nuclear proteins: studies on transplanted B82 cell karyoplast proteins
Authors: Fernig David G. , Mayer R.John
Published: July 2002

Karyoplasts were prepared from B82 cells (thymidine kinase deficient mouse L cells) by cytochalasin B mediated enucleation. Morphological measurements show that the nucleus constitutes 89% of a karyoplast by volume. Homokaryons were obtained by Sendai virus mediated karyoplast‐B82 cell fusion. Transplanted nuclei were not destroyed catastrophically but were maintained intracellularly for at least 140 h. Transplanted nuclear proteins were degraded with an average half‐life of 84 ± 7 h by processes partially sensitive to inhibition by NH4Cl (50%) and leupeptin (30%). The data therefore suggest that some nuclear proteins are translocated to the cytoplasm for lysosomal degradation.
Tetrameric alkaline phosphatase from human liver is converted to dimers by phosphatidylinositol phospholipase C
Authors: Hawrylak K. , Stinson R.A.
Published: July 2002

Membrane‐bound human liver alkaline phosphatase solubilized by a non‐ionic detergent, Nonidet P‐40 (NP‐40), has the molecular mass of a tetramer. It can be converted to a dimeric form by treatment with a phosphatidylinositol phospholipase C (PI‐PLC) obtained from Bacilluscereus. When human liver plasma membranes were directly treated with PI‐PLC, the released alkaline phosphatase was dimeric. Thus, phosphatidylinositol may help maintain the tetrameric quaternary structure of alkaline phosphatase and aid its binding to human liver plasma membranes.
Synthesis of 11,12‐leukotriene A4, 11S,12S‐oxido‐5Z,7E,9E,14Z‐eicosatetraenoic acid, a novel leukotriene of the 12‐lipoxy genase pathway
Authors: Kitamura Shigeto , Shimizu Takao , Izumi Takashi , Seyama Yousuke
Published: July 2002

A simple and efficient method for preparing 11,12‐leukotriene A4 has been established by the stereospecific biomimetic route from arachidonic acid. 12S‐Hydroperoxy‐5Z,8Z,10E,14Z‐eicosatetraenoic acid was synthesized using a partially purified 12‐lipoxygenase of porcine leukocytes. The methyl ester of the compound was then chemically converted to two labile epoxides with a conjugated triene structure. These compounds were identified by proton NMR and mass spectrometry to be 11S,12S‐oxido‐5Z,7E,9E,14Z‐eicosatetraenoic acid (11,12‐leukotriene A4) and its geometric isomer.
Biosynthesis and intracellular transport of glyoxysomal malate dehydrogenase in germinating pumpkin cotyledons
Authors: Yamaguchi Junji , Mori Hitoshi , Nishimura Mikio
Published: July 2002

Glyoxysomal malate dehydrogenase was synthesized as a larger molecular mass precursor in germinating pumpkin cotyledons. In pulse‐chase experiments, the radioactive larger molecular mass precursor (38 kDa) disappeared and was converted to the mature form (33 kDa) of the enzyme. When the radiolabeled cotyledon was fractionated into cytosolic and organellar fractions, the larger molecular mass precursor was first recovered in the cytosolic fraction and then only after a 20 min chase the mature form was found in the organellar fraction. This indicates that the higher molecular mass precursor is synthesized in the cytosol and the processing of the transient precursor is coupled to the transport into glyoxysomes.
The cytoskeletal protein vinculin is acylated by myristic acid
Authors: Kellie S. , Wigglesworth N.M.
Published: July 2002

In non‐muscle cells the mechanism by which microfilament bundles interact with the plasma membrane is unclear. Vinculin, a 130 kDa protein found in adhesion plaques, has been postulated to have a role as a membrane anchor for microfilaments and we have investigated the biochemistry of this molecule in more detail. We report that a fraction of vinculin in chick embryo fibroblasts is acylated by myristic acid. This modification was present in both membrane‐bound, cytoskeletal and cytosolic vinculin and thus did not determine preferential subcellular localisation. Myristic acid was also present in vinculin from cells transformed by Rous sarcoma virus.
Catalytic activities of synthetic octadeoxyribonucleotides as coenzymes of poly(ADP‐ribose) polymerase and the identification of a new enzyme inhibitory site
Authors: Hakam Alaeddin , McLick Jerome , Buki Kalman , Kun Ernest
Published: July 2002

The catalytic activity of highly purified poly(ADP‐ribose) polymerase was determined at constant NAD+ concentration and varying concentrations of sDNA or synthetic octadeoxyribonucleotides of differing composition. The coenzymic activities of deoxyribonucleotides were compared in two ways: (i) graphic presentation of the activation of poly(ADP‐ribose) polymerase in the presence of a large concentration range of deoxyribonucleotides and (ii) by calculating kD
values for the deoxyribonucleotides. As determined by method i, auto‐mono‐ADP‐ribosylation of the enzyme protein at 25 nM NAD+ was maximally activated at 1:1 octamer/enzyme molar ratios by the octadeoxyribonucleotide derived from the regulatory region of SV40 DNA (duplex C). At a 0.4:1 sDNA/enzyme ratio, sDNA was the most active coenzyme for monoADP‐ribosylation. At 200 μM NAD+, resulting in polymer synthesis and with histones as secondary polymer acceptors, duplex C was the most active coenzyme, and the octamer containing the steroid hormone receptor binding consensus sequence of DNA was a close second, whereas sDNA exhibited an anomalous biphasic kinetics. sDNA was effective on mono‐ ADP‐ribosylation at a concentration 150–200‐times lower than on polymer formation. When comparison of deoxyribonucleotides was based on method ii (kD
values), by far the most efficiently binding coenzyme for both mono and polymer synthesis was sDNA, followed by duplex C, with (dA‐dT)8 exhibiting the weakest binding. The synthetic molecule 6‐amino‐1,2‐benzopyrone (6‐aminocoumarin) competitively inhibited the coenzymic function of synthetic octadeoxyribonucleotides at constant concentration of NAD+, identifying a new inhibitory site of poly(ADP‐ribose) polymerase.
Covalently bound pyruvate in phosphopantothenoylcysteine decarboxylase from horse liver
Authors: Scandurra R. , Politi L. , Santoro L. , Consalvi V.
Published: July 2002

Horse liver phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) incorporates nonexchangeable tritium from borotritide with a decrease of the activity. Substrate prevents both tritium incorporation and the decrease in activity. Acid and base hydrolysis of the tritiated protein releases labeled lactate identified by highvoltage paper electrophoresis, paper chromatography and silicic acid chromatography. These results indicate the presence of pyruvate covalently bound through an ester linkage to phosphopantothenoylcysteine decarboxylase which is then another example of a mammalian enzyme in which pyruvate is involved in a catalytic activity.
Tetrazole analogs of amino acids as constituents of modifiers of carboxypeptidase A catalysis
Authors: Kojro Elzbieta , Willhardt Ingo , Römbach Annelotte , Grzonka Zbigniew , Hermann Peter
Published: July 2002

The action of substrate analogs containing the tetrazolyl group instead of the C‐terminal carboxy group on the peptidase activity of carboxypeptidase A is studied. The analogs compete with the substrate for the secondary binding site thus showing activation phenomena.
ncreased 25‐hydroxyvitamin D levels in portal blood following cholecystokinin injection in the dog
Authors: Meyer M.S. , Alon R. , Shibolet S.
Published: July 2002

To establish whether an enterohepatic circulation of the metabolites of vitamin D exists, polyethylene catheters were cannulated into the portal vein of dogs. The dogs were then starved for 24 h and injected with cholecystokinin (CCK) to induce gall bladder contraction. At various time intervals thereafter blood samples were collected from the portal and the saphena veins, and sera prepared and analyzed for the metabolites of vitamin D. The serum levels of 25‐hydroxyvitamin D [25(OH)D] were found to be significantly higher in the portal blood when compared with levels in peripheral blood following CCK injection. Since portal blood collects nutrients absorbed from the gut and as the dogs were starved for 24 h prior to blood collection, the only source of the increased concentrations of 25(OH)D in portal blood is likely to be bile. These findings support the notion that an enterohepatic circulation of 25(OH)D does exist under normal physiological conditions.
DNA‐mediated gene transfer as an indicator of DNA damage and its repair by recipient cells
Authors: Ireland Christine M. , Stewart Bernard W.
Published: July 2002

Preparations of plasmid containing the thymidine kinase gene (pHSV106) were treated with the alkylating agents methyl methanesulphonate or N‐methyl‐N‐nitrosourea prior to transfection into thymidine kinase‐deficient mouse L‐cells using the DNA‐calcium phosphate co‐precipitation technique. Relative to transfection with unmodified plasmid, a reduced transformation efficiency was observed using alkylation‐damaged plasmid, N‐methyl‐N‐nitrosourea causing the greatest inhibition. Treatment of recipient cells with arabinosyl cytosine or dideoxythymidine during the expression period following transfection by the ‘damaged’ plasmid reduced transformation efficiency, suggesting that DNA repair 4–6 h post‐transfection was required for gene expression.
High levels of protein carboxyl methyltransferase in well‐differentiated human endometrial carcinoma
Authors: Ben-Baruch Gilad , Solomon Roya , Menczer Joseph , Kloog Yoel
Published: July 2002

The levels of protein carboxyl methyltransferase, an enzyme that methylates free carboxyl groups of proteins, were determined in normal human proliferative and secretory endometrium and in poorly and well‐differentiated endometrial carcinoma. Protein carboxyl methyltransferase activity was 67% higher in the well‐differentiated carcinoma than in the normal tissues, while similar enzyme levels were observed in the two normal tissues and in the poorly differentiated carcinoma. The results suggest that in the well‐differentiated tumor there is excessive protein methylation and offer a possible biochemical probe for distinguishing between the two types of tumor.
Effects of glucose on insulin release and 86Rb permeability in cultured neonatal and adult rat islets
Authors: Boschero A.Carlos , Tombaccini Donatella , Atwater Illani
Published: July 2002

Glucose‐induced insulin release and modifications in 86Rb outflow were studied in cultured neonatal and adult rat islets. The dose‐response curve for neonatal islets was steeper than for adult islets and the maximal response was clearly shifted towards lower glucose concentrations. In neonatal islets, glucose‐induced insulin release was inhibited by the Ca2+‐channel blocker, nifedipine. In the absence of glucose, the 86Rb outflow from neonatal islets was lower than from adult islets. Also, the glucose‐induced reduction in 86Rb outflow was less pronounced in neonatal islets. Altered K+ permeability in the B‐cell membrane could explain the change in glucose sensitivity of neonatal islets.
High propeller twist and unusual hydrogen bonding patterns from the MD simulation of (dG)6 · (dC)6
Authors: Zielinski Theresa Julia , Shibata Masayuki , Rein Robert
Published: July 2002

A molecular dynamics (MD) study of (dG)6 · (dC)6 including counter ions and 292 water molecules was made. The hydrogen bonding pattern and propeller twist angles for the mini‐helix are reported as averages for times spanning 21–30, 31–40, 41–50, and 51–60 ps. The propeller twist angles range from 18° to 38°. Bifurcated and interstrand neighboring base (twisted) hydrogen bonding patterns were found.
The duck β‐globin gene cluster contains a single enhancer element
Authors: Kretsovali A. , Marcaud L. , Huesca M. , Scherrer K.
Published: July 2002

An erythroid‐specific enhancer was previously identified in the 3′‐flanking region of the β adult gene in chicken and duck, by transfection into AEV transformed chicken erythroblasts. Here we show that the duck enhancer is equally active in erythroid human K562 cells, presenting an embryonic/fetal program of globin gene expression. Furthermore, no other enhancer was found within the 20 kb of DNA including four β‐like globin genes as well as a 1.5 kb upstream and a 3 kb downstream sequence.
Refinement of the solution structure of the RNA‐DNA hybrid 5′‐[r(GCA)d(TGC)]2 Combined use of nuclear magnetic resonance and restrained molecular dynamics
Authors: Happ Claudia Scalfi , Happ Erwin , Clore G.Marius , Gronenborn Angela M.
Published: July 2002

The solution conformation of the self‐complementary RNA‐DNA hybrid hexamer 5′‐[r(GCA)d(TGC)]2 is investigated by NMR spectroscopy and restrained molecular dynamics. The 1H‐NMR spectrum is assigned in a sequential manner using two‐dimensional homonuclear Hartmann‐Hahn and nuclear Overhauser enhancement spectroscopy. From the latter a set of 178 approximate interproton distance restraints are determined and used as the basis of a structure refinement by restrained molecular dynamics. Eight independent calculations are carried out, four from a classical A‐type geometry and four from a classical B‐type one. Convergence is achieved to very similar A‐type structures with an average atomic root mean square difference between them of 1.0±0.2 Å. The converged structures exhibit variations in helical parameters similar to those found previously for the analogue RNA hexamer 5′‐r(GCAUGC)2 [(1988) Biochemistry 27, 1735–1743].
A N‐acetyllactosamine‐specific cell‐binding activity in a plant pathogen, Erwinia rhapontici
Authors: Korhonen Timo K. , Haahtela Kielo , Pirkola Anna , Parkkinen Jaakko
Published: July 2002

A strain of the phytopathogenic bacterial species, Erwinia rhapontici, was found to cause hemagglutination of human erythrocytes that was specifically inhibited by β‐galactosides. Of the monosaccharides tested, N‐acetyl galactosamine and galactose efficiently inhibited the hemagglutination. The most potent inhibitor identified was Galβ1—4GlcNAc that was 30–100‐fold more potent than Galβ1—3GlcNac or Galβ1—3GalNAc. Fetuin had no effect on the hemagglutination whereas asialofetuin was inhibitory. No blood group specificity was found for the hemagglutinin. These findings indicate that the E. rhapontici strain possesses a novel bacterial cell‐binding activity with specificity for terminal N‐acetyllactosamine residues.
Different phosphorylated forms of an insulin‐sensitive glycosylphosphatidylinositol from rat hepatocytes
Authors: Merida Isabel , Corrales Fernando J. , Clemente Rosa , Ruiz-Albusac Juan M. , Villalba Mayte , Mato Jose M.
Published: July 2002

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol‐specific phospholipase C from Bacillus cereus. By Dowex AG1 × 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP‐dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP‐dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 × 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin‐sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.
Electron microscopy of glycogen degrading enzymes
Authors: Scraba Douglas G. , Bradley Roger D. , Fitzgerald Paula M.D. , Madsen Neil B.
Published: July 2002

Single molecules of glycogen phosphorylase b exhibit images in the electron microscope which are similar in shape and dimension to those derived from X‐ray crystallography. Phosphorylase a exhibits tetramers but shows dimers in the presence of glucose. Glycogen debranching enzyme appears as a monomer with an unusual crescent or shrimp‐like shape, with occasional isologous aggregation to circular dimers. The longest dimension of the monomer is very similar to that of the phosphorylase dimer, 11.5 nm. Strong binding of the debranching enzyme to glycogen is readily visualized in the electron microscope. It is suggested that the distinctive shape of the debranching enzyme may be related to its catalytic function.
Phosphorylation and dephosphorylation of hormone‐sensitive lipase Interactions between the regulatory and basal phosphorylation sites
Authors: Olsson Håkan , Belfrage Per
Published: July 2002

Phosphorylation of the basal site with glycogen synthase kinase‐4 enhanced the rate of phosphorylation of the regulatory site by cyclic AMP‐dependent protein kinase 1.7‐fold. In contrast, the phosphorylation state of the regulatory site did not affect the rate of phosphorylation of the basal site with glycogen synthase kinase‐4. The rate of dephosphorylation of either the regulatory or the basal phosphorylation site by protein phosphatase‐1, 2A or 2C was independent of the phosphorylation state of the other site. These results suggest that the basal phosphorylation site could play an indirect role in the control of the hormone‐sensitive lipase activity in the adipocyte by functioning as a recognition site for the cyclic AMP‐dependent protein kinase in the phosphorylation of the activity‐controlling regulatory phosphorylation site in response to lipolytic hormones.
Immunochemical characterization of the modulator protein of the ATP,Mg‐dependent protein phosphatase
Authors: Abeele Carline Vanden , Vandenheede Jackie R. , Merlevede Wilfried
Published: July 2002

Polyclonal antibodies raised against the modulator protein of the ATP,Mg‐dependent protein phosphatase completely neutralize all known properties of the purified modulator: inhibition or inactivation of the phosphatase catalytic subunit as well as the kinase FA‐mediated activation of the ATP,Mg‐dependent phosphatase. They do not cross‐react with phosphoinhibitor‐1 or the phosphatase catalytic subunit. Direct analysis of boiled or unboiled skeletal muscle extracts by Western blotting reveals a 32 kDa polypeptide corresponding to the modulator protein as the most dominant protein staining band.
Purification and characterisation of two forms of toxin B produced byClostridium difficile
Authors: Torres Javier F. , Lo¨nnroth Ivar
Published: July 2002

Toxin B fromClostridium difficile was purified to homogeneity by gel filtration and high resolution ion exchange chromatography. Two forms of toxin B were found. Form 1 which seemed to consist of two identical subunits of 220–300 kDa; femtogram amounts of this toxin induced rounding of fibroblast cells. Form 2 contained subunits of 43 kDa and 105 kDa; the stoichiometric ratio probably being 4 : 1; picogram amounts were needed to induce rounding of fibroblast cells. Immunological studies suggested that both subunit types were antigenic and had epitopes which were identical with those of form 1.
Proton NMR resonance assignments and surface accessibility of tryptophan residues of a dimeric phospholipase A2 from Trimeresurus flavoviridis
Authors: Endo Toshiya , Oya Masanao , Kaptein Robert , Vuister Geerten W. , Kihara Hiroshi , Mohri Noriko , Tanaka Shuji , Ohno Motonori
Published: July 2002

Proton NMR spectra of a dimeric phospholipase A2 from Trimeresurus flavoviridis have been recorded. N‐1 proton resonances of the tryptophan indole rings have been detected and assigned to specific positions, Trp‐3/Trp‐30, Trp‐68 and Trp‐108, by comparing the spectra of the enzyme derivatives with tryptophans oxidized to differing extents. Photo‐CIDNP experiments have revealed that Trp‐68 and Trp‐108 are exposed while Trp‐3 and Trp‐30 are buried in the molecule. This is consistent with the X‐ray crystal structure of a homologous phospholipase A2 from Crotalus atrox where residues 3 and 30 are located at a dimer interface, but inconsistent with the results of stepwise oxidation of tryptophan residues.
Evidence that insulin increases the proportion of polysomes that are bound to the cytoskeleton in 3T3 fibroblasts
Authors: Hesketh John E. , Pryme Ian F.
Published: July 2002

The association of polysome redistribution with changes in protein synthesis was investigated in insulin‐stimulated fibroblasts. Free polysomes were released by Nonidet‐P40 and 25 mM KCl, cytoskeletal‐bound polysomes were retained at 25 mM KCl but released at 130 mM, while membrane‐bound polysomes were released by deoxycholate. Insulin increased the proportion of polysomes which were retained at 25 mM KCl but had no effect when extraction was carried out at 130 mM KCl, suggesting that more polysomes were associated with the cytoskeleton. Insulin also reduced the amount of actin released from the detergent‐insoluble cytoskeleton indicating that the hormone affects microfilament organization.
Does sphingomyelin inhibit the erythrocyte anion transport system?
Authors: Scheuring Uwe , Haase Winfried , Schubert Dieter
Published: July 2002

The anion transport protein of the human erythrocyte membrane, band 3, was incorporated into unilamellar sphingomyelin vesicles. The vesicles showed a rapid sulfate efflux which could be inhibited by specific inhibitors of the erythrocyte anion transport system. All band 3 molecules contributing to the inhibitor‐sensitive flux component were arranged ‘right‐side‐out’. The turnover number of the transport protein for sulfate transport was virtually identical to that in phosphatidylcholine bilayers and around 6 times larger than in human erythrocyte membranes. Thus, in contrast to other claims, sphingomyelin does not inhibit the erythrocyte anion transport system.
Ribosomal proteins S2, S6, S10, S14, S15 and S25 are localized on the surface of mammalian 40 S subunits and stabilize their conformation A study with immobilized trypsin
Authors: Marion Marie-Jeanne , Marion Christian
Published: July 2002

Trypsin immobilized on collagen membranes has been used to digest accessible ribosomal proteins of rat liver 40 S subunits. Six proteins (S2, S6, S10, S14, S15 and S25) have been found to be highly exposed on the surface of 40 S particles. They appear to be in close physical contact and localized in the same region of the subunit, most likely protruding at its surface. Electric birefringence reveals that digestion of these proteins results in unfolding of subunits: the birefringence of 40 S particles becomes negative, like that of RNA, the relaxation time undergoes a 15‐fold decrease and the mechanism of orientation is drastically modified.